Nutrient limitation of litter- associated microbes in boreal streams in northern Sweden
An experimental study on nutrients limiting fungal respiration
Madelene Fridell
Student
Degree Thesis in Biology 15 ECTS Bachelor’s Level
Report passed: 12 June 2015 Supervisor: Ryan Sponseller
Abstract
The aim of this study is to evaluate the relative importance of N and P as limiting factor to aquatic fungi growing on leaves in boreal streams in northern Sweden. The study focused on aquatic hyphomycetes (fungi) growing on Betula pendula litter in enclosed containers within a climate controlled room. Water for experiments was collected from five different streams spanning a range of nutrient and total organic carbon (TOC) concentrations and each site had replicate containers with either added nitrogen, phosphorous, nitrogen + phosphorous or ambient. At the end of a three-week incubation period, litter was removed, and microbial activity measured in terms of respiration on leaf discs. In all containers there was a decrease in total N and almost all of the experimentally added inorganic N was consumed by the end of the experiment. In N+P containers most of the added P was used, but only a smaller fraction was used in only P treatments. Concentrations of TOC increased during the experimental period in all containers. There were no significant differences in leaf disc respiration between sites (F=1.483, p=0.571 or sites*treatment (F=2.588, p=0.245). There were, however, significant differences between nutrient treatments (F=4.711, p=0.004).When performing multiple comparisons between different nutrient treatments there was a significant difference in respiration between NP and ambient (p= 0.003) and NP and P (p=0.002) treatments. There were close to significant differences in respiration rate between N and P (p=0.053) and N and ambient (p=0.08) treatments. Overall, results from this experimental study suggest that N is the key limiting nutrient for aquatic hyphomycetes in the system.
However, it is not only the availability of nutrients that affect the respiration and decomposition by aquatic microbes such as aquatic hyphomycetes, there are also other factors such as temperature, pH, macroinvertebrates and other environmental factors.
Key words: Aquatic hyphomycetes, boreal streams, nutrients, nutrient limitation
Table of contents
1 Introduction ………
……..1
1.1 Aim of the study ……….. 2
2 Material and methods ………. 2
2.1 Study organism ……… 2
2.2 Study site ……… 2
2.3 Nutrient additions ………... 3
2.4 Experimental design and setup ……….… 3
2.5 Microbial respiration measurements ………... 4
2.6 Statistical analyses ………... 4
3 Results ……… 5
3.1 Nutrient additions ………. 5
3.2 Respiration ……….. 6
4 Discussion ……….... 8
4.1 Respiration ……… 8
4.2 Nutrients ……… 9
4.3 A changing environment ……….. 9
4.3.1
Clear-cuts………. 9
4.3.2
Nutrient enrichment and nutrient pollution……….. 10
4.3.3
Climate change……… 10
4.4 DOC ……… 11
4.5 Changes in water quality ……….……… 11
4.6 This experiment and future research ……….. 11
4.7 Conclusions ……… 12
5 References ……….. 12
Appendix
……… 151
1. Introduction
Small forested streams are often heterotrophic, meaning that heterotrophic respiration is higher than the autochthonous primary production can sustain and that they are thus supported by inputs of terrestrial organic matter (OM) (Howarth & Fisher, 1976). The particulate organic matter (e.g., leaf litter) that falls in to a stream is thus an important energy source for aquatic food webs. Much of the OM that falls into streams is leaf litter, entering these ecosystems during autumn in temperate and boreal biomes when the trees shed their leaves (Howarth and Fisher, 1976; Allan, 1995). Aquatic microorganisms, such as aquatic fungi (hyphomycetes) and bacteria play a significant role in transferring the energy from these terrestrial OM input to other organisms in the food web through the process of decomposition (Allan, 1995; Sridhar and Bärlocher, 2000; Gulis and Suberkropp, 2003;
Krauss et al., 2011).
When microorganisms (primarily fungi) start to colonize the leaves, usually within hours (Dang, Gessner and Chauvet, 2007), they increase the nutritional value of the leaves which makes them more palatable for invertebrates to feed on (Sridhar and Bärlocher, 2000; Allan, 1995). The aquatic fungi thus play a critical role in ecosystem functioning by making leaves more palatable for shredding invertebrates. This shredding process also creates finer particles which filter-feeders can then utilize, and ultimately may release inorganic nutrients for other microorganisms to use (Krauss et al., 2011), thereby contributing to nutrient cycling and energy transport in streams (Ferreira et al., 2014).
Aquatic fungi make up the largest part of the total microbial biomass in leaf-litter (Krauss et al., 2011), representing as much as 90 % or more of the total heterotrophic biomass, at least in the early stages of leaf decomposition (Frossard et al., 2012; Krauss et al., 2011). This litter may supply the initial nutrients needed for aquatic hyphomycetes growth but these organisms are also able to sequester nitrogen (N) and phosphorus (P) from the water column (Gulis & Suberkropp, 2003). Indeed, increases in nutrient availability in the water column may then increase the microbial activity, thus enhancing the rate of decomposition (Meyer and Johnson, 1983; Gulis & Suberkropp, 2003; Ferreira et al., 2014). With an increased rate of decomposition of autumn-shed leaves it could mean decreased (or altered) food availability for invertebrates during the following winter and spring seasons (McKie and Malmqvist, 2009; Gulis and Suberkropp, 2003) during which the standing stocks of litter from autumn is largely consumed (Gulis and Suberkropp, 2003).
The availability of nutrients in many freshwater systems has increased during the last decades. Human activities, such as agriculture, inputs of waste water and atmospheric N- deposition contribute to nutrient enrichment of aquatic systems (Ferreira et al., 2014;
Woodward et al., 2012). Aquatic fungi growing on litter have been shown to be limited by either N or P or both (Gulis and Suberkropp, 2003). The research on nutrient limitation in boreal landscapes has led to varying outcomes in different systems. Plants and trees in the terrestrial environment are most often N-limited (Näslund et al., 1998) and also the primary production in lakes has been shown to be N-limited in northern Sweden (Bergström, Jonsson and Jansson, 2008). However, Jansson et al (2001) showed that bacteria in northern Swedish lakes can be both P and N-limited, depending on the concentration of dissolved organic carbon (DOC) in the water. DOC concentration is often closely related to the amount of available nutrients and a lake with high DOC and nutrients concentration shows less effect from nutrient enrichment compared to lakes with low concentrations of DOC and nutrients (Karlsson, Jansson and Jonsson, 2002). However, McKie and Malmqvist (2009) showed in their study on streams in northern Sweden that concentration of phosphate in streams correlates better with litter decomposition than ammonium concentrations which could indicate that the organisms responsible for leaf decomposition, mainly aquatic fungi, could be P-limited.
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1.1 Aim of the study
The aim of this study is to evaluate the relative importance of N and P as limiting factor to aquatic fungi growing on leaves in boreal streams in northern Sweden by measuring the respiration. This was done by measuring respiration rates of aquatic fungi growing on birch leaves in a small containers which received a nutrient treatment by adding N, P, both N and P.
Alternative hypotheses
H1: Respiration is higher in containers with added N than in P and ambient, based on studies of N limitation in boreal streams and lakes (Burrows et al., 201;. Bergström et al., 2008).
N is the primary limiting nutrient.
H2: Respiration in containers with added P is higher than in ambient, hypothesis based on field data showing P could be the limiting factor (McKie and Malmqvist, 2009)
P is the primary limiting nutrient.
H3: Respiration is the highest in containers with added N+P, the limiting effect from both N and is removed (Elser et al., 2007).
Co-limitation by N and P.
2. Material and methods
2.1 Study organisms
Aquatic hyphomycetes spread and colonize new leaves in mainly three different ways; direct contact when leaves touch; hyphal fragments transported to a new site and by asexual conidiospores know as conidia or spores transported by water to a new leaf (Dang, Gessner and Chauvet, 2007). When a conidia land on a leaf it rapidly germinates and colonizes the leaf before it starts to produce and release conidia again (Dang, Gessner and Chauvet, 2007).
Up to 8 spores per microgram detrital dry mass can be released per day (Krauss et al., 2011;
Sridhar and Bärlocher, 2000). The freshwater hyphomycetes are a polyphyletic group and consist of both Ascomycetes and some Basidiomycetes and Zygomycetes (Gulis, Suberkropp and Rosemond, 2008). The definition aquatic hyphomycete is based on their ability ”to sporulate under water and thrive on deciduous leaves decaying in streams and rivers”
(Krauss et al., 2011). Their conidia which are adapted to running waters are multiradiate branched or sigmoid shaped and often unpigmented. Many species have sticky mucilage on the tips to attach to substrates (Krauss et al., 2011).
Warty birch (Betula pendula) is a fairly common tree species in the riparian zone at the sites used in the experiment. In the process of decomposing leaves, the litter quality and nutritional content are important in terms of lignin concentrations and C:N ratio within the leaves, since some fungal species prefer leaves with low lignin content to leaves with higher lignin content (Gulis, Suberkropp and Rosemond, 2008). The birch used in this experiment is categorized as a woody species, and the leaves have a half-life of approximately 100 days (Allan, 1995).
2.2 Study site
For this experiment 5 sites were chosen from within the Krycklan catchment which is a tributary to Vindeln River where monitoring and research is performed. The catchment is
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characterized by a mix of forests, lakes and wetlands in different proportions. For a specified view of catchment specifics see table 1.
Table 1. Site characteristics for the five different streams used in the experiment, all situated in the Krycklan catchment (Laudon et al., 2011; Burrows et al., 2015)
Site Name Forest (%) Wetland (%) Lake (%) Area (km2)
C1 Risbäcken 98 2 0 0.48
C2 Västrabäcken 100 0 0 0.12
C6 Stortjärnbäcken 72 25 3 1.10
C7 Kallkälsbäcken 84 16 0 0.47
Vin Vindeln 2 88 12 0 2.35
2.3 Nutrient additions
Background nutrient concentrations in the streams used in the experiment have been frequently monitored and usually the streams have concentrations of nitrogen varying between 10-50 ug/l and phosphorous usually has a concentration around 5-10 ug/l. In order to get an excessive amount of nutrients available to the aquatic hyphomycetes during the experiment the concentrations were substantially increased. Target concentrations for nitrogen were 1000 ug/l and phosphorous were 500 ug/l. To achieve these concentrations sodium nitrate (NaNO3) was used to increase N concentrations and potassium dihydrogen phosphate (KH2PO4) to increase P concentrations. N treatments got 0.03 grams of sodium nitrate per 5 liter of stream water. P treatments got 0.01 grams of potassium dihydrogen phosphate per 5 liter of stream water. N+P treatments got both 0.03 g of NaNO3 and 0.01 g of KH2PO4 per 5 liter of stream water. The added N and P was well mixed with the water before adding 0.8 liter to each container. A water sample was taken from each stream and treatment in order to obtainthe starting nutrient concentrations and total organic carbon (TOC) concentrations.
2.4 Experimental setup
20 liters of water was collected from each of the five sites together with leaves from within the stream (wet leaves). The wet leaves were put in a fine meshed bag in an oxygenated bucket together with 60 grams of the pre-weighed dry birch leaves (Betula pendula). The buckets from each site with both dry and wet leaves in them were left for 13 days in order to get the dry leaves to leach and get ‘conditioned’, allowing microbes to establish on the dry leaves from each site.
For each site there were 4 different treatments: addition of nitrogen (N), phosphorous (P), or nitrogen + phosphorus (N+P), along with ambient (A) for control. Each treatment was replicated 5 times for each stream site adding up to 20 containers per site and a total of 100 containers (picture 1).
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Picture 1. Some of the 100 oxygenated containers used in the experiment.
After 13 days of conditioning, all containers were filled with 0.8 liters of newly collected and nutrient enriched stream water, except for the ambient containers which only got pure stream water.
The dry leaves were previously weighed and 1 gram of dry weight was on average 15 dry leaves. When the containers were prepared with treated or untreated stream water the 15 leaves were added from the conditioning bucket to their containers.
Each container was oxygenated constantly and all were placed in a dark, climate controlled room with a temperature of 12 degrees Celsius.
The containers were daily supervised for 3 weeks in order to get the microbes to establish.
Approximately 100 ml of distilled water was added to the containers after 9, 15 and 20 days due to evaporation.
2.5 Microbial respiration measurements
The microbial respiration was measured through the reduction in dissolved oxygen over a three hour incubation period. Each container had 20 leaf disks cut out from leaves and placed in an individual falcon tube. O2 saturation (%) and mg dissolved oxygen (DO) per liter were recorded for each container together with the time and temperature. The falcon tube with leaf disks was then filled with water from the containers without getting any air bubbles trapped in the tube. All tubes were put in a dark place for 3 hours for respiration to proceed. After 3 hours the tubes were then taken out and by using the oxygen probe in each tube, oxygen saturation and dissolved oxygen, temperature and time was recorded again. To account for the respiration from the free water (i.e. not the leaf-associated microbes) a separate tube was filled with only water from each site and treatment, where each treatment consisting of 5 replicates were mixed and oxygenated for a few minutes before filling up the tubes. These water-only controls were treated in the same way as described above.
When the respiration was measured and recorded all leaf disks were put out to dry in room temperature for 48 hours and then dried at 60 degrees for 48 hours. All leaf disks were weighed and counted and the result was the total dry leaf mass weight.
2.6 Statistical analyses
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Respiration was calculated by subtracting the second measurement oxygen mg/l measurements (O2end) from the starting values (O2start) to obtain the amount of oxygen consumed in mg/l. The water respiration (Wr) was subtracted to obtain the respiration from the leaf discs only. Respiration was divided by the total dry leaf mass weight (LFDw) (g).
Respiration per dry leaf mass weight was divided by the time passed (Tp) between the oxygen measurements:
Consumed oxygen (mg/l O2 /g dry leaf mass/hour) = ((O2end-O2start)-Wr)/LFDw Tp
The respiration data was tested for normal distribution using Shapiro-Wilks test of normality and it passed the diagnostic test for normal distribution and equal variance. The data was analyzed using IBM SPSS Statistics version 22.0. Data was analyzed using 2-way ANOVAs for treatment and/or site effects on respiration and the post hoc test used was Fisher’s Least Significant Difference (LSD). A result within a 95% confidence interval was considered significant.
3. Results
3.1 Nutrient additions
The nutrient enrichment experiment succeeded with N and NP treated containers showing an increase in total N to approximately 1 mg/l more than background concentrations (range among sites 0.34 mg/l to 1.55 mg/l). By the end of the experiment the concentration of total nitrogen had increased by approximately 0.2 mg/l in all ambient and phosphorus treated containers (ranging 0.11 mg/l to 0.35 mg/l) while all N and NP containers showed a substantial decrease of N in the end compared to the beginning of the study with an average of -0.73 (ranging -0.58 mg/l to -0.84).
When analyzing the inorganic nutrient data separately, nearly 99% of the nitrate (NO3) added in the beginning of the experiment was consumed over a three-week period. This NO3
consumption was similar regardless of whether N was added alone or in combination with P.
Ammonium (NH4) concentrations showed only a minor decrease and even a production in some containers. A large fraction of the phosphate (PO4) experimentally added was consumed during the experiment; however, significantly less P was removed when added alone (74%) when in comparison to the removal observed when added in together with N (94%). A small production of P occurred in containers where P was not experimentally added.
The background concentrations of total organic carbon (TOC) between the sites ranged from 19.0 mg/l to 30.0 mg/l (table 2) when samples were taken at day one. The concentrations of TOC increased during the study in all sites and for all the treatments. When the study ended TOC concentrations had increased to range from 26.3 mg/l to 35.0 mg/l with a percentage increase ranging from 15% to 46%. The average percentage increase per site was C1: 23%, C2:
20%, C6: 44%, C7: 18% and Vin: 41% showing the highest increase for the sites where initial TOC concentrations were the lowest.
Table 2. The concentration of total organic carbon (TOC) in (mg/l) for the different sites and treatments. Both start and end concentrations (mg/l) and the percentage increased. Concentrations are from the first and last day of the experiment. All sites and treatments increased the concentrations of TOC during the experimental period.
TOC (mg/L) Treatment Start End % increase
C 1 N 25.9 31.3 21%
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P 26.9 34.0 26%
N+P 25.6 32.0 25%
Ambient 26.7 32.2 21%
C 2 N 24.7 30.1 22%
P 25.2 29.6 17%
N+P 24.8 30.2 22%
Ambient 24.4 28.9 18%
C 6 N 20.6 29.6 44%
P 19.8 29.0 46%
N+P 20.0 28.8 44%
Ambient 20.0 28.5 42%
C 7 N 29.9 34.5 15%
P 29.2 34.7 19%
N+P 30.0 35.0 17%
Ambient 29.3 34.8 19%
Vin N 19.1 27.3 43%
P 19.0 26.3 38%
N+P 20.0 27.8 39%
Ambient 18.7 27.0 44%
3.2 Respiration
After 13 days of conditioning, and 21 days exposed to water enriched with nutrients the mean respiration per gram of dry leaf mass per hour was the highest (4.23 mg O2/g/h, SE=0.34) in the NP treated containers. The rate was high in N (3.72 mg O2/g/h, SE=0.28) compared to mean respiration rates observed in ambient (3.02 mg O2/g/h, SE=0.26) and P (2.93 mg O2/g/h, SE=0.28) containers (figure 1). The containers treated with only P had a mean respiration similar or even slightly below the ambient containers mean respiration (figure 1).
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Figure 1. Mean respiration (mg O2/gram of dry leafmass/hour) for all five sites and the four treatments, N, P, NP and ambient with +/- 1 SE.
To test if there was any significant interaction between the amount of oxygen consumed between sites and treatments a 2-way ANOVA was performed using respiration as dependent variable and site and treatment as factors and Fisher’s LSD as post hoc test. There were no significant differences in leaf disc respiration between sites (F=1.483, p=0.571) or between sites*treatment (F=2.588, p=0.245) but there were significant differences among nutrient treatments (F=4.711, p=0.004) (Table 3).
Table 3. Test of between subjects effects for respiration/g dry leaf mass/hour in different treatments and at different sites. There was no significant difference between sites or sites*treatments but there is a significant difference between treatments. Significant results in bold text.
Source Type III Sum of
Squares df Mean Square F P
Site 5.931 4 1.483 0.735 0.571
Treatment 28.492 3 9.497 4.711 0.004
Site * Treatment 31.059 12 2.588 1.284 0.245
Further analyzing the treatment effects and performing Fisher’s LSD post hoc test by multiple comparisons between treatments there was a significant difference in respiration between NP and ambient (p= 0.003) and NP and P (p=0.002) treatments (table 4). The respiration rate between N and P (p=0.053) was marginally significant different and there was a close to significant difference between N and ambient (p=0.08) treatments (table 4).
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Table 4. Multiple comparisons between respiration rates between treatments nitrogen (N), phosphorus (P), ambient (A) and nitrogen+phosphorus (NP). Significant or close to significant results in bold text* (Fisher’s LSD post hoc test).
(I) Teatment
(J) Treatment
Mean
Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound
A
N -0.70175 0.401615 0.084 -1.50099 0.09749
NP -1.21468* 0.401615 0.003* -2.01392 -0.41544
P 0.08784 0.401615 0.827 -0.71140 0.88708
N
A 0.70175 0.401615 0.084 -0.09749 1.50099
NP -0.51293 0.401615 0.205 -1.31217 0.28631
P 0.78959 0.401615 0.053 -0.00964 1.58883
NP
A 1.21468* 0.401615 0.003* 0.41544 2.01392
N 0.51293 0.401615 0.205 -0.28631 1.31217
P 1.30252* 0.401615 0.002* 0.50329 2.10176
P
A -0.08784 0.401615 0.827 -0.88708 0.71140
N -0.78959 0.401615 0.053 -1.58883 0.00964
NP -1.30252* 0.401615 0.002* -2.10176 -0.50329
* The difference is significant at the 0.05 level.
4. Discussion
4.1 Respiration
Results from this experiment showed no significant difference in respiration rates between the study streams but a clear treatment effect resulting from nutrient addition. These results supports hypothesis 1 (H1) that N is the primary limiting nutrient to litter-associated microbes (primarily fungi) in these boreal streams. Containers that were experimentally enriched with N had the highest respiration rate, although containers enriched with NP was not significantly different from the N treatments. In addition, nearly all of the experimentally added N was consumed during the experimental period. Also, when N was available, the use of experimentally added P increased when compared to containers where P was added alone.
Hypothesis 2 (H2), that respiration was higher in P containers than in ambient containers, was not supported in this experiment. This result stands in contrast to what McKie and Malmqvist (2009) found in their study, that decomposition of leaves are positively correlated with phosphate and negatively correlated with ammonium.
Results from these experiment are consistent with other recent studies showing N-limitation in streams (Burrows et al., 2015) and lakes (Bergström et al., 2008) of northern Sweden, and across the globe (Elser et al., 2007). However, results from this study do suggest the possibility of co-limitation of fungal activity by N and P in these systems (H3), as the highest rates of respiration in my experiment were observed in the containers treated with both N and P. Elser et al. (2007) discussed the widespread occurrence of co-limitation by N and P, and suggested that this happens when there is a shift between these limiting nutrients. When concentrations of the main limiting nutrient increase, a limitation of the second nutrient is induced and they are therefore co-limiting when shifting between nutrients. This has been shown to occur in terrestrial, marine and freshwater ecosystems (Elser et al., 2007) and in this experiment where the containers with combined N and P had the highest respiration rates supports that there could be a co-limitation in the experimental streams.
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However, this experiment was only conducted over a 3 week period with no change of water in the containers and no adding of additional nutrients during the experiment which could have an effect on the final measurements. The single dose of nutrients added in the beginning was mostly consumed but at what time during the experiment is not known. This rapid removal could explain why the final respiration measurements did not differ as much between treatments as was expected although observed differences were significant.
4.2 Nutrients
Several studies have shown an increased respiration and increased rate of leaf decomposition when both N and P concentrations were higher and an increased decomposition when only N was added and no significant effects on decomposition when concentrations of P increased (Ferreira et al., 2014). Previous studies have shown that streams with low initial concentrations of respond more to P additions than by N additions on decomposition rates and fungal biomass (Grattan and Suberkropp, 2001)
Streams which normally have high concentrations of nutrients are less prone to show an effect when one or more nutrients are added, both with N and P (Woodward et al., 2012). A stream with naturally low concentrations is probably more likely to show an effect on decomposition rates, respiration rates or on sporulation rates from a nutrient addition treatment.
4.3 A changing environment
Changes in land use, input of wastewater, atmospheric N-deposition, agriculture and many more activities alter the accessibility of nutrient in ecosystems and contribute to an enrichment of nutrients in freshwaters. Such changes made in the environment by humans have impacts on ecosystems all over the globe. The consequences of nutrient enrichment in aquatic ecosystem are not exactly known although significant progress has been made. It is difficult to predict how aquatic environments react to changes in land use or nutrient enrichment and how processes in a stream will change. Conley et al. (2009) brings up that nutrient limitation can vary by both spatially and seasonally, especially in eutrophic coastal systems. Also there are differences in catchment characteristics, atmospheric deposition, soil characteristics, vegetation cover and more that affects the aquatic systems in a catchment.
These and other factors vary between different aquatic environments which make predicting general patterns to nutrient enrichment slightly more difficult.
4.3.1 Clear-cuts
In Sweden where the forest is managed and clear-cutting is a widely used method it is certainly interesting to know if the method affects the surface waters in the area. Often clear- cutting is followed by an increase in nutrient runoff into streams and lakes (McKie and Malmqvist, 2009). Palviainen et al. (2013) looked at N, P, carbon (C) and suspended solids (SS) loadings to a stream nearby, after clear-cutting and soil preparations were made in an area in eastern Finland. They concluded that if there was a wide buffer zone to the stream, and the clear-cut was less than 10% of the catchment, there was an impact on the water chemistry but it was minimal (Palviainen et al., 2013). In the larger clear-cut, which was more than 30% of the catchment area, there were increases in N, total organic nitrogen (TON), NO3, PO4 and SS export to the stream for several years after the clear cut was made (Palviainen et al., 2013). This study shows that forest management affects the water chemistry but it depends on catchments characteristics such as slope, soil texture, hydrological flows, vegetation cover and atmospheric deposition loads and more (Palviainen et al., 2013).
Regardless, experimental results from my study suggest that nutrient enrichment from clear- cuttings could accelerate rates of litter decomposition and carbon turnover in boreal streams.
In addition to these nutrient effects in area that have been clear-cut recently the amount of
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broadleaved trees and litter often increases (McKie and Malmqvist, 2009). Since microbes more easily can decompose broadleaf-litter compared to the more common coniferous litter often dominating as OM-input in many streams, the decomposition rate can get higher in these streams (McKie and Malmqvist, 2009).
4.3.2 Nutrient enrichment and nutrient pollution
As mentioned before, production in streams are often supported by input of terrestrial organic matter. Stream ecosystems which are autotrophic are supported by the in-stream production while heterotrophic streams are supported by the input of terrestrial OM (Howarth & Fisher, 1976). In autotrophic streams where autotrophs, like algae, are less dominant it is microbes such as aquatic fungi and other detrivores (insects and macroinvertebrates) that facilitates the usage of the organic matter that falls in the stream and makes it more accessible to the rest of the ecosystem (Rosemond et al., 2015; Allan, 1995;
Howarth & Fisher, 1976). The experiment showed that an increase in nutrients (N and combination of N+P) increases activity of both microbes and other detrivores, which leads to faster processing of the OM and quicker carbon losses from the system, either to the atmosphere as CO2 through respiratory losses or loss by downstream transport (Rosemond et al., 2015). Specifically, Rosemond et al. (2015) showed that N and P additions to streams largely contributed to the carbon losses together with temperature and discharge rates and the streams with added nutrients had about twice the decomposition rate (Rosemond et al., 2015). Nutrient enrichment or nutrient pollution may not only lead to increased decomposition rates but also to decreased decomposition rates (Woodward et al., 2012). In streams where nutrient levels are low, decomposition rates usually are low and at medium nutrient levels the decomposition rates are the highest (Woodward et al., 2012). In polluted waters the decomposition usually decreases, not only because of nutrient pollution but because of the overall degraded environmental conditions such as oxygen depletion, when more sensitive taxa disappears from the system and benthic habitats getting destroyed (Woodward et al., 2012). As my study suggests, addition of nutrient increase respiration rates and is therefore more likely to contribute to increased carbon losses from the system if concentrations of nutrients increases.
4.3.3 Climate change
With climate change there will be increased temperatures and altered precipitation-patterns (Fernandes et al., 2014). According to a study made by Fernandes et al. (2014) in Portugal, an increase in temperature makes aquatic fungi more efficient in the utilization of N, meaning that lower concentrations of N to decompose at the higher rate when the temperature is higher (Fernandes et al., 2014). But as they mention in their study and, this needs to be tested in several other places, with other kinds of leaves, different nutrient treatments and different temperatures etc., given the overall difficulty of predicting the response in aquatic environments.
For northern boreal systems, climate change is predicted to have major effects on the aquatic systems (Tetzlaff et al., 2013). Changes in temperature and precipitation patterns will affect the water flow, season and intensity and also the quality of the water, in turn affecting processes and services provided by the aquatic systems (Tetzlaff et al., 2013). If increases in temperatures and precipitation in the form of rain it is not only the flow patterns that changes but there will also be changes in vegetation (Tetzlaff et al., 2013). In coniferous regions such as northern Sweden it is likely that more broadleaf vegetation will grow (Tetzlaff et al., 2013) increasing the broadleaved litter in to the streams. Species composition will most likely change when temperatures increase and flow patterns changes, which may in turn affect processes in streams and lakes (Tetzlaff et al., 2013). Although changes like these may differ in time, an insect may disappear from the system quite rapidly while a shift in vegetation is more likely to occur slowly over many years.
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4.4 DOC
The concentration of the total organic carbon (TOC) increased in all containers during the experimental period. The sites with low concentrations at the beginning increased the most during the experimental period.
When looking at aquatic fungi they require more than nutrients such as N and P in order to grow and sporulate. There is also a need for carbon (C) which the fungi get from the leaves.
Aquatic hyphomycetes takes nutrients from the leaf where they are located and use these for building biomass and sporulate and it has been shown that they are able to get N and P from the water (Gulis and Suberkropp, 2003). Is it possible that the fungi also can get carbon from the water? In a microcosm study there was no effect on the decomposition of leaves although the microbial activity increased which points to a usage of a secondary carbon source (Schlief and Mutz, 2007). This indicates that aquatic hyphomycetes are able to utilize DOC from the water column and therefore the quality of the DOC is most likely important.
The quality and bioavailability of the DOC depends partly on the origin and differ between wetland and forested catchments (Berggren, Laudon and Jansson, 2007). In catchments where there are a large proportion of wetlands, the DOC in the stream water is mostly derived from within the wetlands during baseflow (Laudon et al., 2011). Organic carbon (OC) derived from wetlands generally consists of more recalcitrant carbon compounds (Berggren, Laudon and Jansson, 2007) and is therefore less available to aquatic fungi. However, I found no difference in respiration rates across streams than span in gradients of both DOC and labile DOC, suggesting the chemical variables were not influencing fungal communities. More research on the influence of DOC and DOC quality on aquatic fungi are needed to better understand litter decomposition processes in boreal streams.
4.5 Changes in water quality
As the leaves are decomposing they release nutrients in to the water which can be utilized by the suspended microbes or used again by the microbes on the leaves (Howarth and Fisher, 1976). There is more that can affect the decomposition in a stream than the availability of both organic and inorganic nutrients such as temperature, macroinvertebrates, pH, pollutants, metals etc. Temperature and macroinvertebrates was controlled during the study since they were placed in a climate room at constant temperature and there were no macroinvertebrates present except for in 4 containers by accident.
As time passed during the experimental period pH and other water quality parameters may have changed. As pH was not measured it is difficult to say if there were any changes but it is likely that there were some. Previous studies have shown that aquatic hyphomycetes can grow at a wide range of pH (Suberkropp, 2001). In contrast, other studies showed a increase in leaf decomposition as pH increased while other still show a greater decomposition rate in streams with pH lower than 6 compared to streams with pH above 7,5 (Suberkropp, 2001).
Even though fungi can tolerate a wide range of pH, in comparing a high and a low pH stream, decomposition was higher in streams with higher pH (Suberkropp, 2001). It is uncertain if the containers used in this study changed in pH during the experimental period and what the effects would be if it did.
4.6 This experiment and future research
This study was performed to give some indication on what nutrient can be limiting for aquatic hyphomycetes in streams in this region. There were several problems with the experimental setup if the goal was to see a similar result in a microcosm study as in nature.
This experimental setup with leaves in a container does not imitate the natural environment very well since there is water constantly overflowing and renewed with nutrients and oxygen in nature. The oxygenation in a small plastic container is not enough to imitate a constant overflow. The water was never renewed during the study but distilled water was added to
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compensate for evaporation. DOC concentrations and quality change over time, and also pH might change over time compared to a natural environment. Performing a study in a controlled environment makes it easier to draw conclusion but can create false indications since it is more complicated and more factors affecting the decomposition in the natural environments. The macroinvertebrates and physical abrasion that occurs in streams was not present in the containers which are important in the process of decomposition in leaves.
Some physical abrasion was present when swirling the leaves every day although this is not quite the same as in the stream. In addition, the nutrients added in the beginning of the experiment as a single dose was almost completely used up when the experiment ended.
Future research in this area should focus on doing enrichment experiments in a more natural environment or in a controlled environment with aquaria and actual stream water overflow and look at sporulation rates as well as respiration and measure other parameters, such as pH, DOC quality and how catchment characteristics influences the stream and follow it throughout the year. In addition, estimates of litter biomass change would have added an additional variable to test for nutrient effects. If I would do this experiment again I would have added more nutrients in the beginning or renewed the water with fresh stream water with nutrients added during the experimental period. I would also have measured respiration and chemistry weekly to get a continuous view on the process instead of only beginning and the end, which restricted the interpretation of the results.
4.7 Conclusions
To conclude, results from this experiment suggest that N is primary limiting nutrient to aquatic fungi in theses small boreal streams. There was no effect on respiration rates when only P was added to the containers. Respiration rates were high in containers where only N was added and even higher in containers where both N and P was added. Adding both N and P facilitated a larger use of P in NP containers compared to the usage of P in P containers since. Even though there were differences between containers in respiration rates seemingly depending on nutrient availability there are other parameters that could have affected the results.
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Appendix
Appendix 1. Raw data from measuring oxygen consumption during the experiment
Measuring time, temperature, % dissolved oxygen (DO) and mg/l oxygen. Start (1) and end (2).
Site/treatment Time 1 Temp 1 1% DO 1 mg/l
DO Time 2 Temp 2 2 %DO 2 mg/l DO C1: WATER N 13.27 11,8 101,1 10,95 16.28.00 11,7 99 10,74
C1: WATER P 13.26 11,6 101 10,99 16.27 11,7 99,7 10,82 C1: WATER
N+P 13.28 11,6 101,01 11 16.29 11,7 99,4 10,79
C1: WATER
Amb 13.25 11,5 101,1 11,02 16.26 11,5 99,8 10,86
C1: N 1 13.10.30 11,8 101 10,94 16.12.00 11,5 93,3 10,17 C1: N 2 13.11.30 11,7 100,7 10,93 16.13.00 11,5 91 9,91 C1: N 3 13.12.30 11,7 100,9 10,95 16.15.00 11,4 88,4 9,66 C1: N 4 13.13.30 11,7 100,6 10,92 16.16.30 11,4 88,2 9,64 C1: N 5 13.14.30 11,6 100,9 10,98 16.18.00 11,4 88,9 9,72 C1: P 1 13.05.30 11,6 100,8 10,97 16.05.30 11,5 94,7 10,32 C1: P 2 13.06.30 11,6 100,8 10,96 16.06.30 11,4 94,2 10,29 C1: P 3 13.07.30 11,6 100,7 10,96 16.07.30 11,4 90 9,83 C1: P 4 13.08.30 11,6 100,7 10,96 16.10.00 11,4 89 9,73 C1: P 5 13.09.30 11,5 100,8 10,99 16.11.00 11,4 91,5 10 C1: N+P 1 13.15.30 11,7 100,8 10,94 16.20.00 11,4 90,2 9,86 C1: N+P 2 13.16.30 11,5 100,8 10,99 16.21.00 11,4 93,8 10,25 C1: N+P 3 13.17.30 11,5 100,7 10,97 16.22.30 11,4 87,7 9,58 C1: N+P 4 13.18.30 11,4 100,7 11,03 16.23.30 11,3 85,9 9,41 C1: N+P 5 13.19.30 11,4 100,7 11 16.24.30 11,3 94,3 10,33 C1: Amb 1 13.00 11,3 100,7 11,03 16.00 11,3 97,3 10,66 C1: Amb 2 13.01 11,3 100,8 11,04 16.01 11,5 93,3 10,17 C1: Amb 3 13.02 11,3 100,6 11,03 16.02 11,6 93 10,11 C1: Amb 4 13.03 11,3 100,9 11,08 16.03 11,6 90,3 9,82 C1: Amb 5 13.04 11,2 101 11,09 16.04 11,6 93,2 10,13 C2: WATER N 15.00 11,5 100,6 10,96 18.00 11,1 97,7 10,72 C2: WATER P 15.01 11,3 100,4 11 18.01 11,3 98,6 10,81
C2:WATER
N+P 15.04 11,3 100,3 10,98 18.04 11,4 97,9 10,7
C2: WATER
Amb 15.03 11,3 100,3 10,99 18.03 11,2 97,3 10,67
C2: N 1 14.39.00 11,5 100,4 10,95 17.39 11,6 86,4 9,41 C2: N 2 14.39.30 11,3 100,4 11 17.40 11,6 91,1 9,91 C2: N 3 14.40.30 11,4 100,2 10,94 17.41 11,6 92,6 10,08 C2: N 4 14.41.30 11,3 100,1 10,96 17.42 11,6 90,7 9,88
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C2: N 5 14.42.30 11,3 100,2 10,98 17.44 11,5 89,4 9,72 C2: P 1 14.33.00 11,1 100,2 11,02 17.33.30 11,1 89,6 9,74 C2: P 2 14.34.00 11,2 100,1 10,99 17.35 11,3 91,6 10,04 C2: P 3 14.35.00 11,2 100,4 11,02 17.36 11,5 91,6 9,98 C2: P 4 14.36.00 11,2 100,2 11 17.37 11,7 91,1 9,89 C2: P 5 14.37.00 11,2 100,3 11,02 17.38 11,8 94,3 10,21 C2: N+P 1 14.49.00 11,3 100,1 10,96 17.52 11,2 90,3 9,91 C2: N+P 2 14.50.00 11,3 100,1 10,97 17.53 11,3 95,3 10,42 C2: N+P 3 14.51.00 11,3 100,1 10,99 17.54 11,4 89,7 9,8 C2: N+P 4 14.52.00 11,3 100,1 10,96 17.55.30 11,4 85,5 9,34 C2: N+P 5 14.53.00 11,2 100,1 10,99 17.56.30 11,4 86,9 9,48 C2: Amb 1 14.44.00 11,2 100,1 10,99 17.45 11,3 91,4 10,01 C2: Amb 2 14.45.00 11,1 100,2 11,02 17.46 11,4 89,2 9,74 C2: Amb 3 14.46.00 11,2 100 10,97 17.47 11,5 89 9,7 C2: Amb 4 14.47.00 11,2 100,1 11,03 17.48 11,4 87 9,51 C2: Amb 5 14.48.00 11,2 99,9 10,99 17.50 11,4 91,5 10 C6: WATER N 13.07.00 11,1 100,6 11,06 16.07.00 11,3 97,8 10,71 C6: WATER P 13.09.00 11,1 100,3 11,04 16.09.00 11,4 97 10,61
C6: WATER
N+P 13.10.00 11,3 100,2 10,47 16.10.00 11,5 97,1 10,59 C6: WATER
Amb 13.11.00 11,4 100,2 10,95 16.11.00 11,5 96,6 10,53 C6: N 1 12.49.00 11,3 100,4 11 15.52.00 11,6 94,4 10,25 C6: N 2 12.48.00 11,4 100,3 10,96 15.51 11,77 87,7 9,51 C6: N 3 12.47.00 11,3 100,2 10,98 15.49.30 11,6 93,8 10,19 C6: N 4 12.46.00 11,3 100,3 10,99 15.48.30 11,5 93,7 10,21 C6: N 5 12.45.00 11,3 100,4 11 15.47.30 12,7 88,8 9,43 C6: P 1 12.44.00 11,3 100,5 10,98 15.46.00 11,5 84,8 9,25 C6: P 2 12.43.00 11,3 100,5 11,01 15.44.30 11,3 85,5 9,45 C6: P 3 12.42.00 11,3 100,4 11 15.43.00 11,4 93,2 10,17 C6: P 4 12.40.30 11,2 100,4 11,02 15.42.00 11,1 93,7 10,31 C6: P 5 12.39.30 11,2 100,4 11,02 15.40.30 10,8 90,7 10,04 C6: N+P 1 12.50.00 11,3 100,2 10,95 15.53.00 11,3 81,2 8,9 C6: N+P 2 12.51.00 11,4 100,2 10,95 15.54.00 11,3 88,6 9,7 C6: N+P 3 12.52.00 11,5 100 10,9 15.55 11,3 92,7 10,16 C6: N+P 4 12.52.30 11,6 100,1 10,91 15.56.30 11,2 86 9,55 C6: N+P 5 12.53.30 11,6 100 10,88 15.57.30 11,2 88 9,66 C6: Amb 1 12.57.00 11,4 100 10,92 15.58.30 11,2 90,6 9,95 C6: Amb 2 12.58.00 11,4 100,2 10,95 15.59.30 11,3 90,8 9,92 C6: Amb 3 12.59.00 11,4 100 10,92 16.00.30 11,2 92,2 10,12 C6: Amb 4 13.00.00 11,4 100,4 10,97 16.01.30 11,2 90,6 9,94 C6: Amb 5 13.01.00 11,5 100,1 10,92 16.02.30 11,2 83,2 9,13 C7: WATER N 11.45 10,9 101 11,17 14.45 11,6 99,5 10,82 C7: WATER P 11.43 10,8 101 11,19 14.43 11,6 101,4 11,03 C7: WATER 11.46 10,6 101,1 11,25 14.46 11,6 100,1 10,88
