Effects of application of organic and

inorganic fertilizer on Scots Pine

(Pinus silvestris L.) needle nutrient

composition and tree growth

Anna Bergstedt

Degree Project in Plant Biotechnology and Engineering, 30 hp

Report passed:

Supervisor: Kenneth Sahlén, SLU

1

Preface This master thesis has been performed as a completion of my Master of Science degree in Plant

Biotechnology and Engineering at Umeå University. The study corresponds 30 hp and was performed

in January 2013- June 2013 for Sveaskog AB. I would like to thank my supervisor Kenneth Sahlén,

SLU, for the help during this project. I would also like to thank all personnel at Sveaskog in Piteå for

the pleasant company during breaks and Ann-Britt Edfast for the support.

Anna Bergstedt

June 2013

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Summary This study is included in a large scale project called ”Kolsänkor Norrbotten”, supervised by prof.

Kenneth Sahlén and financed by Sveaskog, LKAB, SLU and SYVAB among others. In the project, 22

different stands in Norrbotten, Sweden, has been chosen for fertilization experiments, where the

intention is to investigate the effects that conventional inorganic and Bio Nutrient fertilizers have on

tree growth, carbon sequestration and needle nutrient composition.

In this study the growth increase and the needle nutrient composition for three of the 22 stands has

been investigated. Each stand was divided into three cohesive parts called Control, Bio Nutrient and

Mineral Nutrient. Bio Nutrient areas were fertilized with one of two doses of Bio Nutrient fertilizer

from SYVAB in 2006, while Mineral Nutrient areas were fertilized with a standard nitrogen-based

inorganic fertilizer in 2006 and 2009. Both fertilizers significantly increased tree growth and the

results were similar for both doses of Bio Nutrient as well as for Mineral Nutrient. Nitrogen

concentrations within the needles increased in response to fertilization and were still higher than at

Control areas in 2012. Generally, there were few differences in nutrient content within the needles at

fertilized areas compared to Control areas.

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Table of Contents Preface ..................................................................................................................................................... 1

Summary ................................................................................................................................................. 2

Introduction ............................................................................................................................................. 5

Purpose and goals ................................................................................................................................ 5

Background ......................................................................................................................................... 5

Fertilization history ......................................................................................................................... 5

Growth response of fertilization ...................................................................................................... 5

Nutrients are necessary .................................................................................................................... 7

Nitrogen flux in trees ....................................................................................................................... 8

Needle growth and respiration......................................................................................................... 9

Tree growth ................................................................................................................................... 10

Objective ........................................................................................................................................... 12

Materials and methods ........................................................................................................................... 13

Experimental design .......................................................................................................................... 13

Fertilization ....................................................................................................................................... 14

Investigations..................................................................................................................................... 16

Tree growth ................................................................................................................................... 16

Needle nutrient content investigations .......................................................................................... 17

Calculations ....................................................................................................................................... 17

Result ..................................................................................................................................................... 19

Biomass increase ............................................................................................................................... 19

Amount of tree biomass................................................................................................................. 19

Tree biomass increase ................................................................................................................... 19

Needle nutrient content ..................................................................................................................... 21

Nutrient/nitrogen ratios ..................................................................................................................... 24

Growth increase versus nitrogen content .......................................................................................... 27

Discussion ............................................................................................................................................. 28

Tree growth ....................................................................................................................................... 28

Needle nitrogen content ..................................................................................................................... 28

Needle nutrient content ..................................................................................................................... 29

Nutrient/nitrogen ratio ....................................................................................................................... 29

Correlation between needle nitrogen content and tree growth .......................................................... 30

Conclusions ........................................................................................................................................... 30

Future prospects .................................................................................................................................... 30

References ............................................................................................................................................. 31

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Appendix 1 ............................................................................................................................................ 33

Protocol for ICP................................................................................................................................. 33

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Introduction

Purpose and goals

The aim of this project has been to investigate how organic and inorganic fertilization has affected the

tree growth and the needle nutrient content in different stands of Scots pine (Pinus silvestris L.). This

has been done by gathering previously collected data and by performing own analyses. The results

have been analyzed and compared between the different stands and treatments, and interpreted with

knowledge about tree growth, nutrient flux and fertilization attained from literature.

Background

The forest industry in Sweden is of great importance to the country’s economy. The trees are used in

many different industries such as the pulp and paper industry, sawmill industry, paper and wood

packaging manufacturing and bio fuel production. In Sweden, there is 22.5 million hectares of

productive forest land. In 2010, 942000 hectares were logged, where almost one fifth was final fellings

and the rest were thinned or cleared areas. In the years of 2006-2010 the annual increment was 103.7

million m3 standing volume (Swedish Statistical Yearbook of Forestry 2012). The Swedish Forest

Agency estimates that the total volume of felled timber in 2011 was 88.8 million m3 standing volume.

The total turnover in 2011 was 216 billion SEK, including 128 billion SEK in export, and the forest

industry employed about 60 000 people (Skogsindustrierna 2012).

Fertilization history

It has been known for long that lack of nitrogen limits growth in northern coniferous forests and

because of this the Swedish forests have been fertilized for many years. Since the middle of 1940 the

Swedish Forest Research Institute has performed extensive research concerning forest nutrition and

fertilization, but experimentation with fertilizers has been performed by agricultural scientist for an

additional century. Before World War II most experiments included applications of different waste

products such as slag from ironwork and wood-ash, since commercial fertilizers were too expensive to

consider in forest growth studies (Tamm et al. 1999). In 1907, when Stockholms Supersulfatbolag,

soon followed by others, started to manufacture nitrogen fertilizers, the usage of commercial fertilizers

could increase. In the 1930s, it was demonstrated that the dispersal of ammonium nitrate in an old

Norway spruce forest in the north of Sweden gave a growth increase (Kardell and Lindkvist 2010).

The same time period a connection between nitrogen fertilization and foliage nitrogen concentrations

was observed by H.L Mitchell and R.F Chandler, and their work was of significance for Swedish

forest research (Tamm et al. 1999). In the end of the 1950s the forest companies started to experiment

with forest fertilization, after consultation with the Swedish Forest Research Institute and in the

beginning of the 1960s it seemed like forest fertilization was both viable and profitable. It had then

been demonstrated that all northern stands had depletion in nitrogen, and that both pine and spruce

could utilize applied nutrients (Kardell and Lindkvist 2010). By the mid- 1970s, approximately

170000 hectares was fertilized annually, but the large doses led to eutrophication of water bodies. The

amount was substantially decreased to about 20000 hectares in the beginning of the 2000s while

current level of fertilized forests have increased to around 70000 hectares, where a normal fertilizer

dose in a pine forest today is 150 kg nitrogen/hectare (ha). This dose is assumed to increase the growth

with about 15-20 m3/ha (Näslund 2013).

Growth response of fertilization

When trees are harvested these days, it is usual that the whole tree is removed, including root and

branches, since every piece could be used in one of the earlier mentioned industries. The complete

harvesting leads to depletion of nutrients in the soil, which affects the growth of new forests. One way

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to cope with the depletion is to return wood ashes to the forest. This distribution will result in no or

little growth increase, though, since wood ashes contain all nutrients except nitrogen (Jan-Erik Liss

2002). Therefore, a nitrogen fertilizer has to be applied as well, at sites with low nitrogen levels.

Conventional fertilizers utilize inorganic nitrogen and are known to increase wood production and tree

growth. After fertilization the nitrogen concentration in the needles increases immediately and reaches

a maximum within a year but cannot be traced after a few years. The rate of wood production starts to

increase after one year and will continue to be affected for about ten years with the maximum wood

production rate after 3-4 years (Fagerström 1977).

The growth response of needle, branch and stem after fertilization is not only depending on

fertilization procedure but also the original nutrient status of the soil. Stands growing on soil with low

nutrient status will generally show a stronger growth response after fertilization (Hyvönen et al. 2008).

It has been shown that nitrogen fertilization causes an increase of nitrogen amino acid concentrations

in pine needles, bark and wood, and it has been proposed that high arginine concentrations in the

needles indicate that the nitrogen uptake is higher than what is needed for tree growth. It can then be

suggested that there is a maximum in how much nitrogen fertilization will increase tree growth

(Nordin et al. 2001). Sometimes growth can be inhibited by lack of other nutrients, commonly

phosphorous and potassium, and the effect of fertilization can therefore be enhanced by addition of

these nutrients, but the original nutrient status of the soil is determining the effect here as well

(Hyvönen et al. 2008). Since there are many parameters affecting how well a stand will respond to

fertilization, such as stand age and soil status, nutrient optimization is an intriguing approach.

Experiments of this kind has been performed on Norway spruce stands in Sweden, to investigate how

much an optimization of nutrient including their proportions can effect growth. The results show a

remarkable increase in biomass growth causing stands to be ready for thinning between 10 and 20

years before the untreated stands (Linder and Bergh 1996). Conventional fertilizers can thus be

optimized and the amount of nutrients can be chosen depending on a specific site in order to maximize

production. To assess the nutritional status within the foliage, the ratio between nutrient and nitrogen

content can be considered. An optimal ratio between each nutrient and nitrogen has been determined

by S. Linder (1995). If a ratio is below this determined value it might indicate that the nutrient is

growth limiting. Because of this knowledge, one can adjust the amounts of each nutrient within the

fertilizer in order to achieve this optimal status within the foliage, which in turn indicates that optimal

internal nutrient status has been attained within the tree (Linder 1995).

Bio Nutrient as an alternative

During the production of conventional inorganic fertilizers the amount of carbon dioxide that is

released is 4 times the amount of produced nitrogen. Bio Nutrient on the other hand, is an organic

fertilizer that is based on digestate of organic material such as sewage sludge or food waste. Hence,

fertilization with a byproduct such as Bio Nutrient could decrease the emissions of carbon dioxide. It

is added to the forest floor in a pelleted or granulated product that contains less than 10% water. Bio

Nutrient contains more than 3% nitrogen and can also be enriched with other nutrients if necessary.

Since Bio Nutrient contains most of the necessary nutrients for trees, which is not the case in

conventional fertilizers, it has a great potential as an economical and environmentally friendly

fertilizer (Sahlén et al. 2009). In organic fertilizers most of the nutrients are organically bound and

because of this, they will become available to the trees successively when the organic matter is

decomposed. Organic fertilizers can therefore be used to attain a long-term fertilizing effect without

any nitrogen leakage to the surrounding environment (Bramryd 2001).

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The usage of Bio Nutrients as a fertilizer could be a step to a more ecologically stable system. What

strengthens the case is that the Swedish government has an assignment related to fertilization with Bio

Nutrients. It is a plan of action to reintroduce phosphorus from different sources, such as digestate of

different organic material including sewage sludge or food waste, to the ground. With this assignment

an impact analysis regarding both health and environmental perspective is demanded

(Miljödepartementet 2012). Therefore it is important to investigate the effects on the trees after

treatment with Bio Nutrient regarding wood production and nutrient content, and also to compare the

results with trees treated with conventional fertilizers.

Nutrients are necessary

There are some elements that have been determined essential for plants, meaning that its absence

causes severe abnormalities in plant growth, development or reproduction (Epsteen and Bloom 2005).

There are two classifications of the essential mineral elements; macronutrients or micronutrients

determined by their relative concentrations in plans tissues. Many experiments have been performed in

order to establish the relationship between different nutrients and forest yield and it is clear that the

essential elements are needed in different amounts. To investigate the nutritional status of a tree it is

common to investigate the nutrients within the foliage, since the foliage of the trees is an accessible

part that includes all different nutrients. After several different trials, a foliage optimal nutritional

status has been defined as specific target needle concentrations for each individual nutrient element

(Linder 1995). The different nutrients are components in different compounds or parts of the plant

cell. The roles of the most important nutrients are briefly described below.

Nitrogen (N)

The mineral element that plants require in largest amounts is nitrogen. It is a constituent of amino

acids, proteins and nucleic acids (DNA and RNA) in all plants and therefore a nitrogen deficiency

quickly hinders plant growth. Nitrogen can be mobilized between needles and therefore growth

limiting levels may not be seen in younger tissues (Taiz and Zeiger 2010). Because of the great

importance of nitrogen, this compound will be discussed further in the coming sections.

Phosphorus (P)

Phosphorus is an important component in plants since it is a compound of DNA, RNA, ATP (carrier

of chemically bound energy) and also the phospholipids that make up membranes. A deficiency in

phosphorous may result in stunted growth and delayed maturation (Taiz and Zeiger 2010).

Potassium (K)

Potassium an activator of many enzymes that are involved in respiration and photosynthesis, and it

also plays a role in the regulation of osmotic potential in plant cells. Potassium can be mobilized

between leafs and needles and deficiency therefore usually shows in more mature tissues (Taiz and

Zeiger 2010).

Calcium (Ca)

Calcium is used during cell division and in the synthesis of new cell walls. Calcium is also required

for the normal functioning of plant membranes and deficiency is never seen (Taiz and Zeiger 2010).

Magnesium (Mg)

Magnesium ions (Mg2+

) are important when enzymes for respiration, photosynthesis and synthesis of

DNA or RNA are about to be activated. It is also a constituent of the chlorophyll molecule. Since this

cation is mobile, deficiency is first spotted in older tissues (Taiz and Zeiger 2010).

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Boron (B)

It has been suggested that boron plays roles in cell cycle regulation, membrane function, nucleic acid

production and cell elongation but the precise function of it is unclear (Taiz and Zeiger 2010).

Depletion in boron may lead to uncompleted needle growth which causes a distorted tree form and

also a decrease in wood production since the needles are not fully effective (Nihlgård et al. 2013).

Nitrogen flux in trees

Accessible nitrogen

The majority of air consists of nitrogen in the form of N2, which in turn is the ultimate source of all

soil nitrogen. But nitrogen does not only enter the soil through rainfall, but also by animal droppings

and remains of dead plants and animals. There are three major steps involved in the turnover of N in

soils. First the larger organic N molecules are degraded by hydrolytic enzymes into smaller organic N

compounds. Second, bacteria and fungi degrade the smaller compounds into amino acids and in the

third step these amino acids can either be taken up directly by the plants or further mineralized to

ammonium (NH4+). Ammonium in turn, can be oxidized in a step called nitrification where the end

product is nitrate (NO3-) (Helmisaari and Helmisaari 1992, Taiz and Zeiger 2010).

The central dogma has been that plants only can access N after mineralization of organic N to

inorganic forms, such as NH4+ and NO3

- has occurred. It has also been claimed that conifers have a

strong preference for NH4+ (Öhlund and Näsholm 2001). Lately it has been increasingly accepted that

several species, among boreal, have the ability to absorb amino acids as well (Näsholm et al. 1998).

Since amino acid N is the most common form of N in soils, this discovery is of great importance. The

cold climate and the acidic soils in boreal forests makes degradation of dead organic matter occur

rather slowly which result in a higher proportion of organic N compounds than inorganic NH4+ and

NO3-, since the two latter have a faster turnover in the soil, where the organic N compounds generally

include different polymers and monomers (Näsholm et al. 1998, Nordin et al. 2001, Lipson and

Näsholm 2001).

Plant solute and nitrogen uptake

Nutrients can be transported to the root or mycorrhizal surfaces either by mass flow or by diffusion.

Mass flow of N in soil water is normally driven by transpiration which causes water movement in the

soil towards the roots. A concentration gradient from the root surface is the driving force in diffusion

of N compounds and can therefore work even at sites with low N concentration. Once the N

compounds are at the surface of the root cells they can enter the root cells. Uptake of all types of soil

N is a concentration-dependent process which is under control of transporters in the plasma

membrane. Since larger molecules like amino acids and ions like NH4+ and NO3

- cannot cross the

membrane passively to get into the cytoplasm, they have to rely on active transport. There are three

different transporters involved in this process (Taiz and Zeiger 2010).

A proton pump, which are also called ATPase, extrudes protons out from the cytoplast which

causes a proton gradient that provide a driving force for transportation of solutes through

channels and carriers.

An ion channel allows simultaneous transport of several molecules along the gradient of one

of the molecules and can transport solutes at a high rate.

Carriers are substrate-specific and are only capable of transporting one solute at a time. When

a specific solute bind to the binding site of the carrier, a conformational change follows which

allow transportation of the solute.

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Once the N compounds have passed the plasma membrane of the root cell it may immediately enter

the symplast by crossing the plasma membrane of an epidermal cell or it can enter the apoplast and

diffuse through the cell walls of the epidermal cells. The solute could either diffuse all the way to the

endodermis via the apoplastic pathway or it could enter the symplast if it is transported across the

plasma membrane of a cortical cell (Figure 1). Once the solute reaches the endodermis it has to enter

the symplast because of the Casparian strip, which is a suberized layer that blocks the entry of water

and solutes into the stele via the apoplast. The solute can then, through the symplastic connections,

pass the endodermis into the stele where it can continue to diffuse from cell to cell until it reaches the

xylem. The solute can then be translocated through the plant via the xylem sap, and because of the

presence of the Casparian strip the solute cannot diffuse back through the apoplast. Thus, a higher

concentration of solutes can be present within the xylem even though the surrounding water consist a

lower concentration (Taiz and Zeiger 2010).

The cations, like arginine, remains charged at the normal pH of the sap and may attach to the xylem

wall, while glutamine is slightly negatively charged or uncharged and will therefore not attach to any

xylem elements. Therefore it might be more efficient to use glutamine in N transport through the

xylem. It has been proposed that the N demand of the whole plant is regulating the N uptake by the

roots and studies indicate that glutamine can be used as a signal to the root to decrease N uptake

(Nordin et al. 2001) Most of the N that the trees take up is used during development of new tissues

with short life-span, such as growth of bark, root hairs, needles and seed formation, while the

developing wood tissues will not bind a lot of N (Lundmark 1986).

Figure 1: The schematic picture describes the types of tissues within the root including the two pathways a solute could take;

apoplastic or symplastic (More et al. 1998).

Needle growth and respiration

In early spring, the buds of Scots pine leaf out and the new shoots grow quickly. During shoot

elongation the photosynthetic production increases rapidly. In late summer the needle growth has

ceased and this is when they reach peak values in photosynthetic production. The following years,

buds are formed at the proximal end of the shoot by early summer, even though the current shoot

proceeds to grow throughout the summer. Most needles of a shoot live for around four years and

hence the pine canopy usually contain four age classes of needles, each with declining photosynthetic

efficiency (Fagerström 1977, Linder and Troeng 1980).

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In Scots pine trees located in Sweden, photosynthesis has its active period during approximately eight

months each year with start in spring when temperatures increase. The low winter temperatures partly

destroy the photosynthetic apparatus within the needles, and during early spring reconstruction take

place. When the water in the soil is no longer frozen, positive net photosynthesis can start to occur,

even if only part of the photosynthetic capacity is restored. The rate of net photosynthesis increases

during summer and is mainly controlled by irradiance, air temperature and the plants access to water.

When the days become shorter and irradiance decrease in the autumn, photosynthesis is limited. But if

the climate is mild, the reduction in photosynthesis does not have to reduce the photosynthetic

capacity. It is not until the water in the needles is frozen that photosynthesis is ceased. During the

period when photosynthesis is active, considerable amounts of carbon is fixed. After growth has

ceased during autumn, the fixed carbon is translocated to the roots where it is stored (Linder and

Troeng 1980).

One can divide respiration into two components; maintenance respiration which supplies energy for a

number of life supporting processes, and growth respiration which supplies energy needed for growth.

Respiration does not only occur via the needles, but also in the tissues outside the cambium of stems

and branches. When comparison of stem growth and patterns of respiration was investigated it was

shown that the maximum rate of diameter increase occurred one month earlier than the peak in

respiration, which could be explained by the fact that secondary and tertiary cell wall-thickening takes

place after the volume increase is finished. This is one of the facts that make it hard to separate growth

respiration from maintenance respiration. It has been shown that the photosynthetic rate per unit

needle is increased when plots are fertilized (Linder and Troeng 1980). An increased N concentration

in the needles causes an increased net photosynthetic rate which leads to a higher growth response, but

there seems to be a time delay before maximum response is attained (Fagerström 1977).

Nitrogen pools in the needles

Nitrogen in the needles can either be mobile or structurally bound and depending on which form it has

during certain time points can lead to different effects. The concentration of available mobile N at the

time of bud formation is determining the following year’s potential production of new needle biomass

but it is the actual amount of mobile nitrogen available at the production of new needles that is

deciding actual needle growth. If the amount of mobile N is not sufficient, needle growth is terminated

earlier than predicted. When the needles are developed mobile N is irreversible immobilized into

structurally bound N (Fagerström 1977)

Nitrogen and other nutrients can be re-translocated between needles, and this happens in one of two

phases. One is during spring and early summer when overwinter storage is moved from all current

needles in different ages to growing tissues such as elongating shoots and needles. During autumn,

prior to needle abscission, the mobile N is withdrawn and transported from the yellowing needles

where the oldest remaining needles are the ones who first receive the nutrient. Because of this latter re-

translocation, N is only drained from the canopy pool through growth, and the remaining needles

during autumn can represent the extent of stored nutrients within the tree. It has been suggested that

the growth rate of a tree that is the main factor controlling re-translocation of nutrients (Fagerström

1977, Helmisaari 1995).

Tree growth

All living things generally strive for two things, to grow and reproduce. In order to grow, a tree needs

to be able to perform photosynthesis and therefore biomass production will be focused on

photosynthetic tissue i.e. needles. When allocation to the foliage is enhanced, the photosynthetic

productive surface increases, but with more foliage the tree needs more mechanical support and

11

improved water and nutrient uptake. Therefore the tree will also focus on biomass allocated to stems,

branches and roots. Wood is not only used as mechanical support and as a transporter of water, carbon

and nutrients, it is also used in storage. The roots are necessary since they attach the tree to the ground

and they also provide the tree with water and nutrients. The tree will prioritize to develop whatever

part that needs to be enhanced if there is any competition, and this is a fact that needs to be considered

when one wants to optimize the yield of wood (Mencuccini et al.1997).

Photosynthesis and respiration

In the needles of conifers both photosynthesis and respiration occur. The photosynthetic reaction

transpires within the chloroplasts of the needle cells where the first step is a light reaction within the

electron transport chain, which creates energy in the form of ATP and NADPH. Then this energy is

used by the Calvin cycle, which is located in the stroma of the chloroplast, to convert carbon dioxide

and water into carbohydrates that can be used for growth and energy storing. Respiration starts with a

process called glycolysis which takes place in the cytosol of all cells. In the glycolysis sucrose can be

converted into pyruvate which releases less than a quarter of the energy stored within the sucrose. In

order to get access to the remaining stored energy the pyruvate molecules are, in the presence of

oxygen, converted into acetyl-CoA which can then enter the Krebs cycle. This process can also be

referred to as the Citric acid cycle and takes place on the inside of the mitochondrial matrix. There, in

an eight-step process involving several enzymes, each acetyl-CoA molecule will cause the formation

of ATP, NADH and FADH2. NADH can then start a chain reaction in an election transport chain

which is located within the membrane that surrounds the matrix. This will produce ATP which

functions as a source of energy for cellular activities (Taiz and Zeiger 2010).

Wood cell types

There are two types of vascular tissues in plants; xylem and phloem, where the xylem is the main

water-conductive element and the phloem is the sugar-conducting element in gymnosperms. Conifer

stems mainly consist of xylem tissue and this is the part that is considered as wood, where the phloem

is the innermost part of the bark. The wood cells are mainly tracheids which are used both in support

and water transport. Before any water transport can occur the cells first have to die by apoptosis,

programmed cell death. All xylem and phloem cell types are derived from one of two cambial mother

cell types called either fusiform initials or ray initials. The fusiform cells elongates vertically while ray

cells elongates horizontally. When an initial cell divides into a phloem or xylem cell, division occur

transverse. After division the cell starts to differentiate but no elongation will occur. Therefore it is the

size of the initial that decides how long the cell will be. The cell can then mature and deposition of the

secondary cell wall will start. The walls become thicker and the fiber chemistry is decided.

Lignification also occur which makes the cell ridged and strong. After that, cell death takes place and

the cell can then start to transport water, but how long the cell will live is dependent on the season,

where cells live longer during the summer. The young and outermost xylem tissue is called sapwood

and it is responsible for both water conduction and mechanical support. Older xylem tissue is called

heartwood and is only providing mechanical support to the tree. The ray cells in conifers are most

commonly ray parenchyma and they serve as radial pathways between the phloem and xylem. They

stay alive even after maturity and play a role in heartwood formation, transport and storage of

assimilates, and are also connected to a variety of processes linked to wounding (Taiz and Zeiger

2012, Mencuccini et al. 1997, Barnard et al. 2013).

Nitrogen within the wood

It has been seen in cross sections that sapwood and inner bark contains a higher proportion of the total

nitrogen than heartwood and outer bark, when three hardwoods and two softwoods were investigated.

The highest nitrogen concentration was seen in the annual rings closest to the cambium with a gradual

12

decrease across the sapwood, where in some of the cases an abrupt decrease was observed in the

transition zone to heartwood. This gradual diminution in the sapwood was associated with the death of

ray parenchyma cells. Apparently, the nitrogen in their cytoplasm is retrieved for reuse somewhere

else within the tree when the parenchyma cells die (Merrill and Cowling 1966). It has been shown in a

field trial that the concentration of total nitrogen in the cambial region was slightly higher in fertilized

than untreated stands of 50-year old Scots pine trees. The concentration of nitrogen in all trees was

lower in June and July than it was in August, and it was also somewhat higher in the top of the stem

than in the bottom (Sundberg et al. 1993).

Nitrogen fertilization appears to affect the width of the annual rings, where the effect seems to be

highest the third or fourth year after fertilization. When slow-release fertilizers are used the growth

increase is more sustained and therefore it is more likely that the wood produced is more uniform in

quality than a large short-term growth increase does (Saarsalmi and Mälkönen 2001). It is the amount

of latewood that mainly influences the basic density of conifers. When a tree is fertilized with nitrogen

the formation of latewood is delayed, giving the thin-walled earlywood cells a longer growth period

before the thick-walled latewood cells starts to grow. Since there will be more earlywood in a

fertilized tree, the annual rings will be more dense (Sundberg et al. 1993, Saarsalmi and Mälkönen

2001).

The availability of nitrogen may affect wood production directly, but it is generally believed that it

increases crown development which in turn increases the amount of photosynthates and auxin which

are needed for growth. Auxin is a plant hormone where the most common naturally occurring one is

indole-3-acetic acid (IAA). IAA is produced in young leaves and is the transported basipetally through

the tree where it affects cambial cell division and wood cell differentiation. The photosynthetic

reaction within the needles also produces sucrose which is the main transport carbohydrate in conifers.

Sucrose is transported through the phloem from the crown along the stem where it supports wood

production. It has been shown that the sucrose gradient is steepest when wood production is highest,

but it seems like it is the activity of the sink and not the availability of sucrose that determines the

carbon allocation (Sundberg et al. 1993, Aloni 2007). An earlier hypothesis was that latewood

formation is induced by a declining IAA concentration, but Sundberg et al. has demonstrated that the

IAA concentration increased in the same period as latewood formation was begun. Interestingly,

several studies performed by Sundberg has showed that the cambial IAA concentration seems to be at

its lowest during the most active period of wood formation in Scots pine. Still, it has been established

that exogenous sucrose and IAA does stimulate tracheid production in conifers (Sundberg et al. 1993,

Sundberg et al. 2000).

Objective

This study is included in the project ”Kolsänkor Norrbotten”, supervised by prof. Kenneth Sahlén. In

the project, 22 different stands in Norrbotten, Sweden, has been chosen for fertilization experiments,

where the intention is to investigate the effects that Mineral Nutrient and Bio Nutrient fertilizers have

on tree growth and carbon sequestration. Since the needle nutrient composition has been analyzed as

well, the fertilization effect on the needle composition can be evaluated, as well as the potential

connection between nutritional status and tree growth. If a connection is found, an analysis of the

nutritional status of the foliage could be a tool for the assessment of expected growth effect, after

fertilization. In this study the growth increase for three of the 22 stands has been calculated, the needle

nutrient composition for the same stands has been analyzed, and the possible connection between both

has been evaluated.

13

Materials and methods

Experimental design

Three stands of Scots pine on different locations in Norrbotten, Sweden, were selected for this trial

(Table 1). The stands were located at similar latitude and close to each other. The altitude for Lill-

furuberget was smaller than for the other sites. Stand age for Hällberget was only 19 years while the

other two stands was about three times as old. The stands were of mesic soil moisture class and the

vegetation was of lingonberry-type. Every stand was divided into three cohesive parts called Control

(C), Bio Nutrient and Mineral Nutrient (a standard nitrogen-based inorganic fertilizer), named after the

treatment that was going to take place. The parts were selected so that they would be as similar as

possible and would have comparable site quality and tree age. The borders were marked and sampling

plots were selected randomly. Each sampling plot was 20 meters in the direction of the service road

and 20-24 meters in width, which gave a total sampling area of 400-480 m2. Every tree that was alive

within the sampling plot was marked with an X-sign on the trunk of the tree at chest-height (1.3 m),

where all trees of chest-height diameter of more than one cm was marked. Trees positioned at the

border of a sampling plot were considered within the plot if more than half its diameter was within the

zone. The true GPS-coordinates of each sampling plot was registered and numbered. The different

stands including the sectioning with its sampling plots are seen in Figure 2.

Table 1: Locations and ages of each stand.

Figure 2: Maps showing the three investigated sites including the areas of each treatment and sampling plots (marked as

yellow dots). The read areas are treated with Mineral Nutrient fertilizer, the green areas are Control and the blue areas are

treated with Bio Nutrient fertilizer (Sahlén 2012).

Name X-coordinate Y-coordinate Latitude, ° Altitude, m Age of stand

in 2006

Lill-furuberget 1800200 7399300 66,54 95 55

Hällberget 1788500 7406600 66,62 264 19

Näverberget 1792700 7415500 66,69 254 64

14

Table 2: Number of sample plots for each site and treatment.

Fertilization

Between the 10th of August and the 8

th of September in 2006, the three stands were treated with Bio

Nutrient fertilizer dose A (BioA), Bio Nutrient fertilizer dose B (BioB) and a standard nitrogen-based

inorganic fertilizer called Mineral Nutrient (M). The two fertilizers have different nutrient contents

(Table 2). The M fertilizer does not contain any heavy metals, synthetic organic substances or organic

materials. The levels of heavy metals and synthetic organic substances within the Bio fertilizer are

below recommended limits for wood ashes reintroduced on forest lands. The Bio Nutrient fertilizer

was a product from SYVAB (Figure 3) and applied in dosages of 500-620 kg N/ha for dose A, and

33% more for dose B (Table 3). It was obtained from sludge that had been used to extract biogas at

Himmelfjärdsvärket. The Mineral Nutrient fertilizer was from SkogCan and it was applied in amounts

of 130-180 kg N/ha. The autumn of 2009, the Mineral Nutrient fertilization was repeated with 150 kg

N/ha. Control sites were not treated at all. Different dosages of each fertilizer were applied and the

total nitrogen dosage thus varied between treatments and locations (Table 4). From now on, Lill-

furuberget will be called Furuberget.

Figure 3: Bio Nutrient from SYVAB, size demonstrated by match (modified from Sahlén 2012).

Table 3: The nutrient content within the two different fertilizers.

Site Control Bio Nutrient A Bio Nutrient B Mineral

Lill-furuberget 8 7 7 9

Hällberget 8 8 8 9

Näverberget 8 7 7 9

Total 24 22 22 27

Fertilizer ds O.M pH N NH4-

N

P K Ca Mg S B

% % % % % mg/kg mg/kg mg/kg mg/kg mg/kg

SYVAB 92.3 61 6.8 4.2 0.46 3.2 2100 2200 3400 - -

SkogCan - - - 27 13.5 0 0 50000 24000 0 2000

15

Table 4: The amount applied fertilizer, including the total nitrogen, in each dose at each location.

In all locations, the added nitrogen quantities were about 2 times as high in BioA fertilized areas as M

fertilized areas, while the amount was between 2.5 to 2.9 times as high in areas fertilized with BioB

than in areas fertilized with M fertilizer (Table 5). The dosages for Bio fertilizers have deliberately

been chosen a lot higher than realistic levels. This in order to enable an assessment of the risks of any

unacceptable negative environmental effects, and that with great safety margins.

Table 5: The nitrogen ratio calculated between the one of two dosages of Bio Nutrient and mineral Fertilizer, in the different

locations.

Nitrogen ratio Furuberget Hällberget Näverberget

Bio Nutrient A/Mineral Nutrient 2.19 1.92 1.87

Bio Nutrient B/Mineral Nutrient 2.91 2.56 2.48

The fertilization was performed by a forwarder with a fertilizer unit attached which spreads the

fertilizer towards the back and to the sides (Figure 4). The spreader can contain about 7 m3 of fertilizer

and in the bottom part of the container there is a conveyor that moves backwards in an adjustable

speed which transports the fertilizer to an opening in the back where it falls down on rotating plates

that throws the fertilizer. The dosage can be regulated through alteration of the speed of the conveyor

and the width can be adjusted by changing the speed of the rotating plates. SkogCan fertilizer was

spread from every other thinning road as is custom while fertilization with Bio Nutrient was

performed from every road and by several runs, since that dosage was much higher. For dose A the

distribution was divided in three runs and for dose B it was divided in four runs.

Furuberget Hällberget Näverberget

kg ds/ha kg N/ha kg ds/ha kg N/ha kg ds/ha kg N/ha

Bio Nutrient A 14700 617 13400 562 14500 609

Bio Nutrient B 19500 821 17800 747 19300 810

Mineral Nutrient 488+555 132+150 522+555 142+150 653+555 176+150

Mineral Nutrient in

total

1043 282 1077 292 1208 326

16

Figure 4: A) Bio Nutrient fertilizer is placed in the spreader. B) The rotating plates that throws the fertilizer. C) Investigation

of how evenly the fertilizer has been spread. D) Collection bag used for determining how well the fertilizer was spread. E)

The amount of Bio Nutrient spread when the dose was 14 ton/ha. F) The route performed by the spreader at Näverberget,

recorded by GPS, where the Mineral Nutrient was spread to the left and Bio Nutrient to the right. The middle part is the

untreated Control site (Sahlén 2012).

Investigations

Tree growth

The trunk diameter in mm was measured at the already marked position, one (in 2007), three (in 2009)

and six (in 2012) years after fertilization. No biomass data at Hällberget for BioA and BioB areas in

2012 were measured because of time limitations. By using the trunk diameter and the biomass

function for Scots pine as independent variables, the total dry weight biomass for all measured trees

could be calculated. The above ground biomass was calculated with Marklunds biomass functions

(Marklund 1988) while the below ground biomass was approximated by functions described by

Peterson and Ståhl (2007). The probable foliage increase at fertilized sites has not been considered

when biomass was calculated. This data was received and tree growth has then been calculated as

biomass increase (tons/ha and percentage) for the time periods 2007-2009, 2009-2012 and 2007-2012.

The result has also been calculated to show the annual biomass increase in percentage, to make the

results for the different locations and treatments more comparable.

17

Needle nutrient content investigations

Sampling of needles was performed after two (autumn 2008) and six (autumn 2012) years. When

sampling was executed, all needles from one zone were pooled into one sample named after the

number of sampling plot and treatment. The needles were sampled with a pole pruner from the upper

third of the crown and from one year old shoots. They were then dried at 85 °C for 48 hours before

they were cleaned and minced. The needles sampled in 2008 were sent to University of Helsinki where

they were analyzed and following nutrients were determined: C, N, P, K, Ca, Mg, B, Al, Cd, Cu, Fe,

Mn, Si and Zn. Carbon and nitrogen was determined by a combustion method using a varioMax CN

from Elementar Analysensysteme GmbH in Germany while the other nutrients was determined by an

analytical tool called inductively coupled plasma/optical emission spectrometry (ICP/OES), Thermo

Scientific icap 6000 series. Only results for N, P, K, Ca, Mg and B are presented in this report.

The ICP/OES is a powerful tool for the determination of trace elements which is based upon excitation

of atoms and ions that causes spontaneous emission of photons. The samples needs to be in liquid or in

gas phase so the solid ground needle-samples needs to be digested with acid before they are injected

into the instrument. After injection the sample solution is converted into an aerosol that quickly

vaporizes within the 10000K warm core of the ICP. Since the photons have individual energies

depending on the atom or ion that is excited, the origin of an element can be determined by the

measured wavelength of the photons (Hou and Jones 2000). The intensity can then be converted into a

concentration by the usage of a calibration curve.

The needles sampled in 2012 were weighed in and dried at 105 °C for more than 19 h in order to

investigate moisture content. The ICP/OES analyze was performed to determine contents of P, K, Ca,

Mg, S, Fe and Cu in the needle samples (Appendix 1). The samples were also investigated for total

nitrogen. Unfortunately, some problems arose when the ICP analyze was performed. First, two thirds

of the samples were accidentally diluted with a solution containing additional potassium, followed by

an investigation with the ICP. The mistake was observed and the dilution was performed again

according to the protocol. But this time the ICP gave doubtful intensities when looking at the

phosphorus and copper results, while all other values seemed accurate. Since a reference-sample was

run four times each trial, with little variance in both trials respectively, it could be used to assess the

accuracy of the results. Therefore the P and Cu results from the first run were used together with the

K, Ca, Mg and Fe results from the second trial, since the intensities for P and Cu seemed to be

trustworthy in the first trial. As only about two third of all samples were investigated in the first trial,

the dataset including P and Cu is not complete as it does not include more than a few results from

BioA and BioB. By some unknown reason sulfur could not be seen in any samples in any of the trials.

N and C were determined by and Elementar Analyzer called Isotope Ratio Mass Spectrometer and the

analysis was performed by SLU. Combustion of dried C and N sample material converted the samples

to CO2 and N2, and mass spectrometric measurement on CO2 and N2 yield could determine N and C

contents.

Calculations

The data from all different samplings have been compiled to determine the effects the different

fertilizers have had on tree growth and on the needles, in the aspect of nutrient content. As mentioned

the growth increase was calculated. For the nutrient contents, an average for each specific location and

treatment was calculated. The ratio between nutrient content and nitrogen content for each sampling

plot was also calculated, and after this the average ratio for each specific location and treatment was

calculated. With the result for nutrient/nitrogen ratios, the earlier mentioned target value for each ratio

is presented as a dotted line.

18

With MS Excel, two-sided T-tests were performed in order to investigate if the mean value of one

population (a treatment at a location) was significantly different from the mean value of another

population (another treatment at the same location). In order to decide if equal variance or unequal

variance should be chosen when comparing the two datasets, an F-test first needed to be performed,

which showed the two-sided probability that the variance of two datasets were significantly different

from each other. The average deviation within the population was showed with error bars. The growth

increase was plotted against the needle nitrogen content in 2008 in order to investigate if there was a

correlation between nitrogen content and growth increase.

19

Result

Biomass increase

Amount of tree biomass

The average biomass within each location was quite similar between all treatments in 2007 (Table 6).

The average biomass at Hällberget was considerably smaller than for both other locations.

Table 6: Average total dry weight biomass for each measured year.

Tree biomass increase

Total biomass increase was significantly larger at treated areas than at C areas (Figure 5). Exceptions

to this was Furuberget 2009-2012, where the biomass increase for C area was not significantly

different from M area, both at around 10 tons, and at Hällberget in 2007-2009, where the biomass

increase for the C area was not significantly different from the increase at BioA area, both at around 5

tons. At Furuberget the total increase at fertilized areas in time period 2007-2012 was around 20-25

tons, while the increase at Näverberget was close to 30 tons.

Figure 5: The total biomass increase at the different locations, at investigated time periods and different treatments.

Similarities and significant differences (p<0.05) are indicated by letters for each treatment and time period.

Average total biomass in tons/ha 2007 2009 2012

Furuberget Control 66.9 69.0 79.4

Bio Nutrient A 76.7 86.5 102.0

Bio Nutrient B 67.5 76.5 93.3

Mineral Nutrient 62.2 70.5 80.8

Hällberget Control 7.9 11.9 13.3

Bio Nutrient A 11.4 17.6 -

Bio Nutrient B 14.3 22.8 -

Mineral Nutrient 15.3 23.5 37.1

Näverberget Control 73.7 82.1 91.1

Bio Nutrient A 83.3 96.0 114.1

Bio Nutrient B 73.3 84.3 101.1

Mineral Nutrient 77.7 93.0 106.0

20

At Furuberget and Näverberget, the biomass increase in percentage was significantly lower at C areas

than treated areas (Figure 6). An exception to this was Furuberget 2009-2012, where the increase at C

area was not significantly separated from the increase at BioA or M area (Figure 6). At Hällberget, the

increase at C area was not significantly different from fertilized sites, with an increase around 50%, in

the time period 2007-2009. In the time period 2007-2012 for both Furuberget and Näverberget, the

increase at C area was around 20% while the increases for treated areas were between 33-39%.

Figure 6: The total dry weight biomass increase at the different locations, at investigated time periods and different

treatments. Similarities and significant differences (p<0.05) are indicated by letters for each treatment and time period.

At Furuberget and Näverberget, annual biomass increase was similar between fertilized areas and was

generally between 6-8% per year (Figure 7), while C areas had a lower increase at close to or below

5%. At Hällberget the increase at was between 15-34% for all treatments.

Figure 7: The total dry weight biomass increase each year, at the different locations, at investigated time periods and

different treatments, calculated from the result in Figure 6.

21

Needle nutrient content

The nitrogen content within the needles were significantly higher in autumn 2008 than in autumn 2012

at all fertilized sites, but not at the C sites where the values were similar both years (Figure 8). The

values for fertilized sites are about 0.3-0.4 percentage units higher in 2008 than in 2012, with the

exception of Furuberget treated with M, where the difference between the years was lower.

Figure 8: The average needle nitrogen content in the autumn of 2008 compared to the content in the autumn of 2012.

Similarities and significant differences (p<0.05) are indicated by letters for both years of sampling.

Generally, there was no difference in nitrogen content between the fertilized areas, while the nitrogen

content at C areas were significantly lower at both time points for all locations (Figure 9). At

Hällberget in 2012, there was no significant difference between treatments. In 2008, the general

difference in nitrogen content between C areas and any fertilized area was 0.4-0.8 percentage units

while the difference was lower in 2012, at around 0.4 percentage units.

Figure 9: The average needle nitrogen content in the autumn of 2008 compared to the content in the autumn of 2012.

Similarities and significant differences (p<0.05) are indicated by letters for each treatment and location.

22

Little variation in the nutrient concentrations was seen between treatments at Furuberget (Figure 10a).

Needle P concentration in 2008 was not different between C and M area, or between BioA and BioB

area. The latter had significantly higher P concentration, around 1500mg/kg, than C and M areas,

where it was between 1200-1300 mg/kg. Needle Mg concentration at C area in 2008 was higher than

for the three other treatments, with a value close to 850 mg/kg. Needles of fertilized areas contained

about 650 mg/kg. Concentrations of K and Ca did not differ between treatments in 2008, where K

concentrations were around 4500 mg/kg and Ca concentrations were close to 3500 mg/kg. In 2012,

BioA and BioB needle P concentrations were still not significantly different from each other but the

concentrations were lower than in 2008. Needle Ca concentrations at C area was significantly lower

than for BioB, and the concentration at BioB area was also higher than at M area but not separated

from BioA. Mg concentration at C area in 2012 was similar to the concentration in 2008, but with no

significant difference from M. Concentrations at BioA and BioB areas was around 1100 mg/kg and

significantly higher than concentration at C and M areas.

At Hällberget, needle P concentrations in 2008 and 2012 were not significantly different between C

and M areas, or between BioA and BioB areas, but the latter were significantly higher than at C and M

areas (Figure 10b). Concentrations for 2008 were close to values at Furuberget the same year, while

concentrations in 2012 were lower for all treatments. In 2008, the K concentration for M area was

around 4300 mg/kg and significantly lower than all at other areas but BioB. The same year, Ca

concentration for BioB and M areas were significantly different from each other but they were not

different from BioA or C areas. M area had the lowest Ca concentration at around 3000 mg/kg. Mg

concentrations at M area was significantly lower than all other treatments, at around 500 mg/kg. In

2012, the K concentration at BioA and BioB sites were both around 1500 mg/kg and not significantly

different from each other, but all other comparisons were. The concentration of K at M sites was close

to 3200 mg/kg and C areas it was above 4000 mg/kg. The Ca concentration at C area were higher than

the fertilized areas and had a value close to 4000 mg/kg. Ca concentration at M area was almost 3000

mg/kg and significantly larger than only BioA, while BioA and BioB areas had concentrations just

above 2000 mg/kg. The concentration of Mg at BioA and BioB areas was around 500 mg/kg while C

and M areas had concentrations above 800 mg/kg. There was no difference in Mg concentration

between C and M areas, or between BioA and BioB areas, but the latter were significantly lower than

C and M.

At Näverberget in 2008, the P concentration at the C area was around 1000 mg/kg and significantly

lower than at fertilized sites (Figure 10c). P concentration at BioA area was around 1400 mg/kg and

significantly higher than M. The K concentration at the C area was almost 3600 mg/kg but was

significantly lower than at BioA and M areas, while fertilized areas were not significantly different

from each other. The Ca concentration at C area was around 2500 mg/kg and significanlty different

from the concentration at BioA and BioB areas. The Ca-concentration at M area was at around 2700

mg/kg and not significantly different from BioA. The concenration at BioA area was in turn not

different from concentration at BioB area either, both with Ca-concentrations around 3200 mg/kg. For

the Mg concentration, a similar result as the one at Furuberget in 2008 was aschieved. In 2012, the

result for the concentration of P was similar to that for Hällberget 2012, while the K result was similar

to the result at Näverberget 2008, with the exception of the K concentration at the M area which was

not higher than at the C area. The Ca concentration at the C area was at around 2900 mg/kg and only

lower than at the BioB area, while the others were not separated. The Mg concentration at BioA area

at about 900 mg/kg was not separated from BioB area, but lower than at C and M areas. All other

concentrations were above 1100 mg/kg and were not significantly different from each other.

23

Generally for all three locations, the P concentration was significantly higher at BioA and BioB areas

than at C and M areas. BioA and BioB P concentrations were not significantly different from each

other in any of the years at any locations. Concentration of P at C and M areas were not significantly

different from each other in any of the years at any locations, except at Näverberget in 2008 when M

area had a higher concentration than C area.

Figure 10: Average needle nutrient content in the autumn of 2008 and 2012 at a) Furuberget, b) Hällberget and c)

Näverberget. The average value of a nutrient was compared between treatments. Similarities and significant differences

(p<0.05) are indicated by letters for each nutrient and treatment.

a

b

c

24

The boron concentration was generally around 30 mg/kg at all locations no matter the treatment, with

only a few exceptions (Figure 11). For Furuberget, the concentration at C area was lower than the

concentration at M area, at around 15 and 33 mg/kg respectively. All other treatments were not

significantly separated from each other. At Hällberget there was no significant difference between

treatments. At Näverberget, the boron concentration at M area was around 50 mg/kg and was not

significantly separated from the concentration at BioB area, but it was higher than at C and BioA

areas. The concentration at BioA area was not significantly different than the concentration at C area,

both close to 29 mg/kg. The concentration at C and BioB areas were not significantly separated.

Figure 11: Average needle boron content in the autumn of 2008. The average value of boron was compared between

treatments. Similarities and significant differences (p<0.05) are indicated by letters for each treatment and location.

Nutrient/nitrogen ratios

There was a large variation of the ratio, between average needle boron content and average needle

nitrogen content for the autumn of 2008, between locations and treatments (Figure 12). At both

Furuberget and Hällberget, the significant difference between treatments were the same as the average

boron content result (Figure 11), but at Näverberget there was a difference. There, the ratio at C area

was not significantly separated from ratio at BioB and M areas. The ratio at BioB area was not

significanlty different to the ratio at BioA area, both with values at around 0.2%, while C and M areas

had values around 0.3%.

Figure 12: Needle boron/nitrogen ratio in the autumn of 2008. The ratio was compared between treatments and target values

are marked with a dotted line. Similarities and significant differences (p<0.05) are indicated by letters for each treatment and

location.

25

All ratios at C areas were higher than ratios at treated sites at Furuberget (Figure 13a). The ratios at C

areas were all above the target value. Ratios for BioA, BioB and M areas were not significantly

different from each other for any of the nutrient/nitrogen ratios in 2008, except the Mg/N ratio, where

the ratio for M area was higher. In 2012, the ratios for P/N at BioA and BioB areas were not

significantly different from each other. The K/N ratio at C area was 35 and only significantly higher

than the ratio at M area, while all other treatments did not differ from each other. The three fertilized

sites showed K/N ratios slightly below the target value. The Ca/N ratio at M area was around 26 and

was significantly lower than the ratio at all other sites. The Mg/N ratio at M area was also lower than

the ratio at the other locations, with a ratio around 7.

At Hällberget in 2008, the P/N result showed that ratio for C area was only similar to ratio at BioA

area, but higher than ratios at BioB and M areas (Figure 13b). The P/N ratio for BioA area was also

similar to the ratio at BioB area. The ratio at M area was significantly lower than ratio at all other

treatments, with a ratio close to 9. For the three other ratios in 2008, C areas had significantly higher

ratios than the other treatments. The K/N ratio at BioA area was higher than at M area with a ratio of

almost 35, while the ratios at BioB and M areas were both around 30 and not significantly separated.

For both Ca/N and Mg/N ratios, BioA and BioB areas were not significantly different at close to 25

and 4 respectively, while ratio at M area was significantly lower than BioA and BioB areas, at around

20 and 3 respectively. All ratios at C areas and most ratios at fertilized sites were above the target

values. In 2012, the P/N result showed that the ratio at C area was close to 10 and only significantly

lower than the ratio at BioA area. The P/N ratio at BioA, BioB and M areas are all significantly

different from each other. The K/N, Ca/N and Mg/N ratios at C areas were higher than the ratios at

fertilized sites, and the ratio at M area was significantly higher than at BioA and BioB areas. The two

latter were not significantly different from each other. The K/N ratio at M area was slightly lower than

the target value, while the ratio at BioA and BioB areas were less than half the target value.

P/N, K/N and Mg/N ratios at C areas in 2008 were significantly higher than the ratios at treated sites at

Näverberget (Figure 13c). The Ca/N ratio at C area was around 24 and only higher than the ratio at

BioB and M areas, while BioB and M areas had ratios around 17. The ratio at BioA area was not

significantly separated from the ratio of any other treatment. Most results in 2008 were similar to the

results of Furuberget in 2008. In 2012, there was no significant difference between treatments for the

P/N, K/N or Ca/N ratios. The Mg/N ratio at C area was higher than ratio at BioA and BioB areas, but

not higher than at M area. The ratio at M area was not different from ratio at BioA or BioB area. In

2012, all K/N ratios are slightly lower than the target value.

26

Figure 12: Needle nutrient/nitrogen ratio in the autumn of 2008 and 2012 at a) Furuberget, b) Hällberget and c) Näverberget.

Each ratio was compared between treatments and target values are marked with a dotted line. Similarities and significant

differences (p<0.05) are indicated by letters for each ratio and treatment.

a

b

c

27

Growth increase versus nitrogen content

The growth increase between years 2007-2012 is plotted against the needle nitrogen content measured

in 2008 at Furuberget (Figure 13) and Näverberget (Figure 15). At Hällberget, the increase between

years 2007-2009 was used instead, but with the same nitrogen values (Figure 14). At both Furuberget

and Näverberget, the C samples were clearly separated from treated samples which in turn did not

appear to be clearly separated from each other. For both these locations, the growth increase was lower

when the nitrogen levels were lower. The growth result for C area at Hällberget was not separated

from the other treatments, but the nitrogen concentrations were lower at C areas than at fertilized

areas.

Figure 13: The growth increase between years 2007-2012 was plotted against the needle nitrogen content measured in the

autumn of 2008 at Furuberget.

Figure 14: The growth increase between years 2007-2009 was plotted against the needle nitrogen content measured in the

autumn of 2008 at Hällberget.

Figure 15: The growth increase between years 2007-2012 was plotted against the needle nitrogen content measured in the

autumn of 2008 at Näverberget.

28

Discussion Since the ICP analyses were performed by different people and different machines the two different

years, it is hard to compare the results with each other. It is therefore easier to compare how the

different treatments have affected the amount of one element at one time point and also compare the

results from the different locations. Since the nitrogen analyses were performed by qualified people

both times is can be assumed that these results are more comparable. There was a large difference in

total nitrogen application for the M, BioA and BioB treatments at 150, ~600 and ~800 kg N/ha

respectively. Because of this, it is very hard to compare the results directly. If Bio Nutrient fertilizer

had been applied in 150kg N/ha just like Mineral Nutrient, then possible differences in effects on

either nutrient status or tree growth could have been attributed directly to one of the treatments. Now,

differences might be due to either the fertilizer or the large difference in nitrogen addition.

Tree growth

It is obvious that all three fertilizations have significantly increased tree growth, this based on the

result of Furuberget and Näverberget. Since no data concerning BioA or BioB areas was available

from Hällberget after 2009 it is not possible to conclude anything about the effect of Bio Nutrient

fertilizer at this location. At this location there was no significant difference between C and M area. It

is also clear that the annual growth was larger at Hällberget than on the other locations, which is

probably because of the young age of the stand. When considering annual tree growth the result from

2007-2012 is likely the most relevant since that is the longest time period. An obvious difference

between C areas and fertilized areas is seen at both Furuberget and Näverberget, where the annual

growth was approximately 30 % higher at fertilized sites. At these two locations, no significant

difference between the total biomass increase during the period 2007-2012 at the tree fertilized sites

were observed. In fact, the biomass increase was very similar between BioA, BioB and M areas at

both locations. This indicates that for an initial time period of around 5 years, the effects on tree

growth of Mineral fertilization at standard dose and Bio Nutrient fertilization in both doses are similar,

this despite the large difference in total nitrogen application for the three treatments. One possibility is

that the Bio Nutrient fertilization will give a more long term effect due to its organically bound

nitrogen and large dose. Therefore it would be interesting to investigate tree growth at the same

locations at a later stage. Preferably more than 10 years after fertilization, since that is considered as

the longest time span in which effects of Mineral fertilizers can be seen (Fagerström 1977).

Needle nitrogen content

The nitrogen concentration within the needles had increased at all fertilized sites two years after

fertilization, which supports earlier results (Tamm et al. 1999), indicating that nitrogen fertilization

does increase the needle nitrogen content in the years closest to fertilization. At C areas the nitrogen

content was significantly lower than at fertilized sites both in 2008 and in 2012. The concentration of

nitrogen at C areas was also very similar both investigated years which indicates that around 1% of

nitrogen within the foliage is normal conditions for all three sites. Two years after fertilization the

concentration was around 1.4-1.8 % at all fertilized sites, no matter the treatment. No significant

difference between the three treatments were noticed, except at Furuberget where M area had a lower

nitrogen concentration than both Bio Nutrient areas. In 2012, fertilized sites were back to baseline

levels at Hällberget. This was not the case at the other locations, which is interesting. At Furuberget

and Näverberget, the nitrogen content was still significantly higher at fertilized areas than at C areas,

but levels were lower than in 2008. Note that the treated areas were not significantly different from

29

each other. Since needles only live for about four years in Scots Pine, an increase in nitrogen

concentrations should not be seen after this time period if there was no lingering effect of the

fertilization. For the Bio Nutrient treated areas, this result might indicate that there is a long term effect

of the fertilizer. Since Mineral Nutrient was applied again in 2009, at a similar dose as in 2006, an

elevated nitrogen concentration in 2012 should probably be expected due to this. However, if a second

dose of Mineral Nutrient had not been applied, it is probable that the nitrogen content within the

needles at these areas would have returned to baseline levels in 2012. Regarding the second

application of Mineral Nutrition, the nitrogen concentration was investigated three years after

fertilization and not two, which was the case in 2008. If one would have investigated the needle

nitrogen content in 2011instead of 2012, it is possible that similar result in nitrogen content as in 2008

would be seen at M areas.

Needle nutrient content

In general, there seems to be little or no differences between C and fertilizes areas with the result of

phosphorous as an exception. Even though both calcium and magnesium was added at all fertilized

areas, generally no increase in needle concentration was observed. In Bio Nutrient fertilizer potassium

was also included, but fertilization did not result in any clear increase of K concentration within the

needles. The Mineral Nutrient also included boron, and at Furuberget and Näverberget the boron

concentration was higher at M areas than at C areas, but differences between M and Bio areas were in

most cases not significant. It has been shown earlier that nitrogen fertilization can depress levels of

other nutrients within the foliage, and by the addition of these elements along with nitrogen when

fertilizing, a decrease can be prevented (Tamm et al. 1999). Earlier it has also been suggested that the

concentration of an element within the foliage remains constant or decreases when there is an

increased availability, even though growth can increase (Helmisaari and Helmisaari 1992). Based on

this, the achieved result is quite reasonable and no increase should be expected, and this might explain

the decrease in magnesium concentration for fertilized sites at Furuberget and Näverberget in 2008.

When considering the increase of phosphorous concentration at both Bio Nutrient sites, which were

significantly higher than the concentrations at C and M sites at all locations in 2008 and 2012, it can

be suggested that this is a result of the high phosphorous addition at Bio Nutrient fertilized areas. In

contrast, Mineral Nutrient fertilizer did not contain any phosphorous at all, while BioA and BioB

dosages included above 400 and around 600 kg P/ha respectively.

Nutrient/nitrogen ratio

When considering the nutrient/nitrogen ratio result, it should be noticed that the nitrogen content in all

fertilized sites were significantly higher than nitrogen concentration at C areas (except from Hällberget

in 2012), and therefore a ratio below target value at a fertilized site might be because of a high

nitrogen concentration and not because of a low concentration of the nutrient. Both Bio Nutrient

treatments resulted in similar ratios for all nutrients, at all locations and at both time points. Generally

there were no significant differences between BioA, BioB and result for M areas. Only for potassium,

ratios for all fertilized sites were clearly below target value. This result was seen more or less at all

locations and at both time points, and it might indicate that potassium was in fact growth limiting. It is

interesting that there were little or no significant differences between the K/N ratios at Bio and M

areas, even though potassium was included in the Bio Nutrient fertilizer. Perhaps the dose of

potassium should have been larger in order to ensure that the element would not limit growth.

30

Correlation between needle nitrogen content and tree growth

For Hällberget, Control sampling plots were only separated from fertilized areas by nitrogen content

and not by growth increase. At both Furuberget and Näverberget, Control samples were separated

from all fertilized samples both by nitrogen content and by growth increase. The different result at

Hällberget might be because of the young age of the stand when fertilized, and this aspect makes it

hard to compare the location with the others. If only Furuberget and Näverberget are considered, the

result might indicate a relationship between high needle nitrogen concentration and growth increase.

Since it has been established that nitrogen fertilization does increase both tree growth and needle

nitrogen content, it was expected to find some correlation between these two.

Conclusions Generally, C areas had less biomass increase than treated areas (except for Hällberget).

o The performance at fertilized sites at both Furuberget and Näverberget was, in most

cases, not significantly different from each other.

Nitrogen concentrations within the needles increased in response to fertilization and were

higher than at C areas in both 2008 and in 2012 (except for Hällberget in 2012).

There seems to be little or no difference in nutrient contents between Control and fertilized

areas except for phosphorous.

o Phosphorous concentration was higher at Bio Nutrient areas than Control and Mineral

Nutrient areas, in all locations.

Bio Nutrient fertilizer consisted of 3% P while Mineral Nutrient fertilizer did

not contain any P.

Nutrient/nitrogen ratios indicate that no other nutrient except nitrogen was growth limiting,

except perhaps K

A correlation between needle nitrogen content and tree growth was indicated at Furuberget

and at Näverberget.

Future prospects In the project ”Kolsänkor Norrbotten”, 19 other stands have been fertilized in similar ways, one in

2006 and the rest in 2007 and 2008. The results for the sites that were fertilized in 2007 and 2008 will

be analyzed in autumn of 2013 and 2014 respectively. When these results are added to the result

presented in this report, it is possible that more conclusions can be drawn.

It is known that conventional fertilizers can have a positive effect on tree growth for up to 10 years.

Studies have shown that organic fertilizers similar to Bio Nutrient can have a positive effect on tree

growth a lot longer than what conventional fertilizers do (Sahlén et al. 2010). It would therefore be

interesting to investigate these locations again in the future to see if there has been any difference in

tree growth at Bio and M areas. So far, fertilization with Bio Nutrient seems like a sustainable option

that gives the same result in tree growth as conventional fertilizers do. But since different amounts of

nitrogen were added it is hard to assess how big amounts of Bio Nutrient that has to be applied in

order to give the same effect as Mineral nutrient at 150 kg N/ha. This would therefore also be

interesting to investigate in the future.

31

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33

Appendix 1

Protocol for ICP

In the method called “foliar” the mass fraction (ω) of chosen elements within conifer needles can be

investigated. The unit for mass fraction is mg/kg. The procedure is described in following steps:

1. Needles were dried at 70 °C for 24 hours, then grounded and mixed. Samples are stored in

room temperature.

2. Samples were weighed to an amount between 200-300 mg into preparation tubes (mA).

Samples were also weighed and dried at 105°C for more than 19 h in order to investigate how

dry they were (mB).

3. Reagents, 4 ml 7.28 M nitric acid (HNO3) and 1 ml 9.8 M hydrogen peroxide (H2O2) was

added to the preparation tubes.

4. The samples were digested in a microwave (model Mars 5, CEM, program method

Barr091209 – Xpress). The temperature slowly increased during 20 minutes to 180 °C, and

then this temperature was kept for 10 minutes. After the program had run, the tubes had to

cool for at least one hour.

5. Samples were diluted first to a volume of 50 ml in several steps. First 5 ml MQ water was

added to the samples. Then 2 ml of the diluted sample were diluted again to an amount of 10

ml, which gave the total volume of 50 ml or a dilution factor 10. But before samples could be

run through the ICP, they had to be diluted another 10 times, which therefore gave a dilution

factor and a total volume of 100.

6. Samples were pumped into the ICP one by one where they were heated with radio waves,

which caused the different elements do disintegrate into atoms. This lead to an atomic

emission and the different emissions could be seen at different wavelengths depending on the

atom. Since its intensity was proportional to the mass fraction of the specific element, and

since the ICP registers the different intensities (γ), the mass fraction could then be calculated.

The formula used for the calculation is ω = (γ • V)/m where m is the dry weight of mA,

calculated from the result of mB (modified from a protocol written by Anders Ohlsson, version

1.1, 2011-03-09, SLU).