Field application of organic and inorganic fertilizers and ...
Effects of application of organic and inorganic fertilizer ... · Bio Nutrient as an alternative...
Transcript of Effects of application of organic and inorganic fertilizer ... · Bio Nutrient as an alternative...
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
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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
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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
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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.
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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).