Nanotechnology is a fast growing scientific area, and the related industries have rising in the air. Nanomaterials that involved in the nanotechnology are rather small and with unique properties that we have not yet fully understood. In this point of view, we need to gain more knowledge about the influence of nanomaterials to the natural ecology, since we will soon face a lot of “Nano wastes”. The objective of this study is to understanding the influence of ZnO nanoparticles to different fungi, and how it changes their growing competence. Through the results, we can analyze the potential eco-toxicity of the ZnO nanoparticles, which is considered as safe materials when it in the size scale of micrometers or bigger. We selected four fungi and observed their growth under six different ZnO nanoparticle concentrations, and found that the nanoparticles have different effect on the growth of different fungi. Also, we found ZnO nanoparticles can change the balance of two fungi that were growing at the same culture. Based on the results, we discussed three models about how the nanoparticles could be toxic to the forest eco-system, and conclude that there are potential risks if the nanoparticles are explored to the nature.

Keywords: Zinc oxide, Nanoparticles, Nano-ecotoxicity, Fungus

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Acknowledgements

When I started to write this thesis, I realized that I obtained helps from many kind persons.

First of all, I would like to thank Morgan Fröling for his guidance. I would also like to thank Renyun Zhang and Fredrick Carlsson for their helps during the time I working in the lab. I thank Mattias Edman for valuable discussions. I thank Håkan Norborg and Torborg Jansson for their technical helps. I should also thank Prof. Håkan Olin for supporting experimental chemicals.

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List of Figures

Page 2: Figure 1. Example of zinc oxide products, such as paintings, toothpastes, sun protections and bandages.

Page 6: Figure 2. TEM image of as synthesized zinc oxide nanoparticles.

Page 8: Figure 3. Photos of Antrodia serialis grown at different concentrations of ZnO nanoparticles and different days (4, 6, 8, 10, 12 days).

Page 8: Figure 4. The growing curves of Antrodia serialis at different concentration of ZnO nanoparticles.

Page 9: Figure 5. Photos of Skeletocutis odora grown at different concentrations of ZnO nanoparticles and different days (4, 6, 8, 10, 12 days).

Page 9: Figure 6. The growing curves of Skeletocutis odora at different concentration of ZnO nanoparticles.

Page 10: Figure 7. Photos of Phellinus viticola grown at different concentrations of ZnO nanoparticles and different days (4, 6, 8, 10, 12 days).

Page 10: Figure 8. The growing curves of Phellinus viticola at different concentration of ZnO nanoparticles.

Page 11: Figure 9. Photos of Fomitopsis pinicola grown at different concentrations of ZnO nanoparticles and different days (4, 6, 8, 10, 12 days).

Page 11: Figure 10. The growing curves of Fomitopsis pinicola at different concentration of ZnO nanoparticles.

Page 12: Figure 11. Schematic drawing of the size of the fungi after 2, 8 and 14 days growth.

Red lines show the size of the fungi without the presence of ZnO, blue line show the size of the fungi in the presence of ZnO.

Page 14: Figure 12. Photos of fungi after 4 days growth in the presence of ZnO at concentration of 5.

Page 15: Figure 13. Schematic drawing the competition of fungi Phellinus viticola and Fomitopsis pinicola without and with the presence of ZnO nanoparticles. The solid lines indicate the size of original planted fungi, and the dash lines show the possible size and shape of these two fungi after two weeks growth.

Page 16: Fiugre 14. The competition of Phellinus viticola and Fomitopisis pinicola with and without the presence of ZnO.

Page 17: Figure 15. Schematic model of the Eco-system change due to the pollution of ZnO nanoparticles.

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Terminology

ZnO: Zinc oxide

HMT: Hexametylentetramin

TEM: Transmission electron microscope Fungus A: Antrodia serialis Fungus B: Skeletocutis odora Fungus C: Phellinus viticola Fungus D: Fomitopsis pinicola

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1. Introduction

1.1. Project scopes

The main scope of the project is to study the potential eco-toxicity of ZnO nanoparticles. To do this, we selected four fungi that were selected from the forest and investigated the influence of ZnO nanoparticles to their growth at different concentrations. Based on the results we will see how can the nanoparticles inhibit the growth, which might lead to the balance among fungi in nature, resulting eco-toxicity. Competing growth of two fungi in the presence of ZnO nanoparticles will be done to show how it can break the balance between fungi. We will discuss three simple models to analyze the influence of such nanoparticles.

1.2. Background

1.2.1. What is ecotoxicity?

Ecotoxicity is a scientific topic that is to study the field of ecotoxicology (a portmanteau of Ecology and Toxicology) refers to “the potential for biological, chemical or physical stressors to affect ecosystems” [1]. Such stressors could happen in nature when the levels of toxicity are high enough to disrupt the natural behavior and interactions of the living organisms that comprise the ecosystem. There is another word namely environmental toxicity [2], which is also refer “the potential stressors to nature”. However, there are differences between, where ecotoxicology integrates the effects of stressors across all levels of biological organization from the molecular to whole communities and ecosystems, but environmental toxicology is mainly about at the level of the individual [3].

The ultimate goal of ecotoxicology is to protect eco-system from toxic matters [4], so that we are to be able to predict the effects of pollution so that the most efficient and effective action to prevent or remediate any detrimental effect can be identified. In those ecosystems that are already impacted by pollution, ecotoxicological studies can inform as to the best course of action to restore ecosystem services and functions efficiently and effectively.

1.2.2. What is nano-ecotoxicity?

Nano-ecotoxicity is to study of the ecotoxicity of nanomaterials [5]. Nanomaterials are those materials with at least one dimension at the range of 1 to 100 nm [6], which have versatile applications in many commercial products, including tires, stain-resistant clothing, cosmetics, computer chips, drug-delivery systems, medical imaging, and implants. However, despite the already widespread use of nanomaterials in modern technology, the lack of information on the human and environmental implications of manufactured nanomaterials is acute [7].

EU has lunched a project named “Nano-ecotoxicity” that coordinated by Dr Claus Svendsen in the Centre for Ecology & Hydrology at Natural Environment Research Council, UK. The project is to evaluate the effect of zinc oxide and silver nanoparticles on earthworms [8]. The researcher pointed out that accumulated nanoparticles might in the longer-term ultimately become toxic in cell and tissues, which is in according to our idea that the accumulation of zinc oxide may influence the growth of fungi that would later lead to environmental problems [8].

1.2.3. Zinc oxide

Zinc oxide is widely used in our daily life, for instance the cosmetic, the paintings, the cloth and so on [9]. Figure 1 shows some examples of the zinc oxide products. The nanotechnology of zinc oxide is especially developing fast in recent years. Zinc oxide is considered as a safe material that can be dropped without further care. However, the nano sized ZnO could have some biological effects to bacteria and fungi leading to environmental problems [10~13], which has already been demonstrated. In fact, since we have used ZnO in many places, the

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nanoparticles of zinc oxide will end up in waste water or sediment in somewhere in the nature.

However, the impact of the nano particles is barely studied.

Figure 1. Example of zinc oxide products, such as paintings, toothpastes, sun protections and bandages.

1.2.4. Fungi

Fungi are a group of organisms in the ecosystem [14], which plays an important role in the ecosystem. Fungi are special due to the cell walls, which are thicker and can resist the attack from the outside world.

Along with bacteria, Fungi are the major decomposers in most terrestrial (and some aquatic) ecosystems, and therefore play a critical role in biogeochemical cycles and in many food webs [15]. Fungi are mainly decomposers, sitting in they nutrient cycling, especially as saprotrophs and symbionts. The role of fungus is degrading organic matter to inorganic molecules that can be used by other organisms.

1.3. How to integrate zinc oxide nanoparticles and fungi, and how it will interact with environment

1.3.1. Nanomaterials

Nanomaterials are kinds materials with at least one dimension less than 100 nanometers. For example, nanoparticles (NPs) are often defined as particles ranging in diameter from 1 to 100 nm, which is quite small. The size of nanoparticles at such range could enable them with quantum effect, which has wide application potential in such as electronics, medicine, chemistry, and so on. Take Finland for example, the turn over of nanotechnology could achieve 1.2 billion euros [16].

Nanoscience and nanotechnology are fast developing fields in the last 20 years. Nanomaterials have been wildly used in recent years, for example nanomaterials embedded clothes, electronic industries, and so on. However, these nanomaterials could be released to nature during or after the use of the products. And, since the nanomaterials are very small, they could have the possibility to enter cells and even interact with proteins, which might cause the death or

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enhanced growth of some microorganisms, such as bacteria and fungi, leading to environmental problems.

The potential market for nanotechnologies is very big. However, problems might appear when we start to use ”nano” contained products, because these nanomaterials could be released to nature without monitoring and the environmental issues have not been clear investigated.

Besides, the awareness of people to the harmful of nanomaterials to nature is not obvious.

If the nanomaterials were released to nature, the first creature that would react to it could be micro bios, such as bacteria and fungi. The small size of the nanomaterials could enter the bacteria or fungi and interact with, or influence the function of some proteins. The results of these interactions would lead to the death, or inhibited growth, or enhanced growth of the fungi of bacteria, depending on the kinds and properties of the nanomaterials.

1.3.2. Fungi and environment

The bacteria and fungi are the basic indicators of environmental. Fungi are groups of eukaryotic organisms, which include microorganisms and moulds. Fungal cells have cell walls that contain chitin, unlike the cell walls of plants, which contain cellulose. When nanomaterial was attached to the fungus, how the cell walls react, how the fungus react, what are the impact to the fungus, are interesting. If the influences are known, it will be interesting to the future study about the changed-fungus how they interact with the environment, like soil, other animals.

1.3.3. Project aims

This project is aims to study the response of some fungi to nanomaterials: ZnO nanoparticles.

The growth of the fungi will be monitored upon addition of ZnO nanoparticles at different concentration. Based on the results, we are going to analyze the potential eco-toxicity of ZnO nanoparticles.

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2. Methods

In this project, the fungi and ZnO nano particles will be used in the experiment.

The fungi, basidiomycete will be separated from wild categories (by Fredrik).The ZnO nanoparticles will be synthesized in lab (by Ying). The media culture will be prepared in the lab (by Ying).

2.1. Preparation of ZnO nanoparticles

Materials: Zn (NO3)2!6H2O was purchased from MERCK. Hexametylentetramin (HMT) was purchased from KEBOLAB. Ethanol (95%) was purchased from Solveco. Filtermembrane was purchased from MILLOPORE.

Synthesis Processes:

1. Measure 400 ml ethanol in a cylinder

2. Weigh 0.5 g Zn(NO3).6H2O and put it into a glass beaker. Pour 300 ml ethanol into the beaker from the cylinder and boil it.

3. Put 5g HMT and the remain 100 ml ethanol together and stir it and put this into the boiling mixture. After 8 minutes, stop the reaction and filtrate the suspension, leaving the white ZnO nanoparticles on the membrane. Then dry the ZnO sample in an oven at 80°C for 1 hour. Then, we get the ZnO nanoparticles powder ready for usage.

Characterization: The synthesized zinc oxide nanoparticles were characterized by using transmission electron microscope to analyze the size distribution. The TEM experiment was done by Dr. Renyun Zhang.

2.2 Prepare for the media culture.

Ingredients (1.5 liter): All ingredients list below were purchased from Sigma. agar 30g [Merck], Glukos 7.5g [ProLaBO], Malt extract 7.5g [Fluka], NH4NO3 0.75g[CdB], MgSo4 (7H2O)0.75g [Fluka,], KH2PO4 0.75g [Riedel-deHaen]

Processes:

1. Put stir bar into a beaker and put agar in it.

2. Add 1.125 liter water into the bottle and stir for 2 minutes.

3. Put all the ingredients in to the solution and add the 0.375 liter water 4. Stir the solution until all ingredients mix perfectly.

5. Put different volume of zinc oxide nanoparticle solution (0.01g/ml), in the 0 series (e.g.

0A, 0B, 0C, 0D), no zinc oxide was added, in the 1 series (e.g. 1A, 1B, 1C, 1D) , the media culture will be added 4.5 ml zinc oxide solution. In the 2 series (e.g. 2A, 2B, 2C, 2D), the media culture will be added 9 ml zinc oxide solution. In the 3 series (e.g. 3A, 3B, 3C, 3D), 18 ml zinc oxide solution will be added. In 4 series (e.g. 4A, 4B, 4C, 4D), 36 ml solution will be added. In 5 series (e.g. 5A, 5B, 5C, 5D), 72 ml solution will be added.

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6. The mixtures were then autoclaved for 10 min at 121°C, 15-lb pressure. When it cooled down to 55 °C, the cultures were taken out from the antoclave and pour into plastic plates with above 20 ml culture per plate. After 2 hours, the plates were packed in plastic bags and sealed with tapes. The packed bags were stored in a cooling room before use.

2.3. Fungi preparation

This part of work was done by Dr. Fredrik Carlsson, who prepared the mother plants. It took 2 weeks for the mother plants to grow.

Antrodia serialis, three replicates, abbreviated as Fungus A1, A2 and A3 in the text.

Skeletocutis odora. three replicates, abbreviated as Fungus B1, B2 and B3 in the text.

Phellinus viticola, three replicates, abbreviated as Fungus C1, C2 and C3 in the text.

Fomitopsis pinicola, three replicates, abbreviated as Fungus D1, D2 and D3 in the text.

2.4. Fungi transplanting

The fungi were transplanted from the mother plant following the procedure as follow:

A metal tube with diameter of 1 cm was first immersed in 70% ethanol for 5 min, and then burn the ethanol on a fire to remove the ethanol and kill bacteria on the tube. After it was cooled down to room temperature, press the tube on the mother plant and carefully take the mother plant with the tube and place it on new plate. Mark and seal the plastic plates with parafilm carefully. After all plates were sealed, they were put in a box in dark at room temperature for growth.

The plates were marked following the rules:

For example, 1A1-1, the first 1 is the concentration of zinc oxide ranking from 0 to 5 as described in section 2.2., A1 is replicate of fungus ranking from A1 to A3, and the last 1 is the replicates number in my experiment that ranks from 1 to 3.

In total the number of samples is, 6 (concentrations) x 3 (mother plant replicates) x 3 (replicates) x 4 (different fungi) = 216.

2.5. Observation and measurement

The sizes of the 1 cm transplanted fungi (all 216 plated) were measured every second day with a ruler for two weeks. Meanwhile digital images were taken. The recorded data were later plotted to show the growth of the fungi.

2.6. Competition of fungi

I did this part together with Renyun Zhang, because it requires more advanced experimental technique that need long time training.

In this part, all the plates’ preparation were the same as above. When doing transplants, two different fungi (Phellinus viticola and Fomitopsis pinicola) were planted in the same plates.

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3. Results

3.1. Synthesized zinc oxide

Figure 2 shows the transmission electron microscope (TEM) image of as synthesized ZnO nanoparticles. By statistically calculating the size distribution of the diameters of the nanoparticles, we found that the average size is 18 nm. The shapes of the nanoparticles were not regular, which is typical for ZnO nanoparticles at this size range.

Figure 2. TEM image of as synthesized zinc oxide nanoparticles.

3.2. Growth of Fungi at different conditions

3.2.1. Species A–Antrodia serialis

Figure 3 shows photos of Antrodia serialis that grown at different concentrations of ZnO nanoparticles. Results indicated that the colonies of the fungi grew bigger when it was in the culture for extending days. However, when the concentrations of ZnO nanoparticles increased, the growth speed was inhibited, as we can see the higher ZnO concentration, the smaller the colonies.

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Figure 4 shows the growing curves of the fungus in 14 days. At concentration levels of 0, 1 and 2, there is not significant different for the growth of Antrodia serialis. However, when the concentration went high, the growth were inhibited, as we can see the growth at concentration 5 could just reach the 60% of concentration 0.

3.2.2. Species B–Skeletocutis odora

Different from Antrodia serialis, Skeletocutis odora was found more sensitive to the presence of ZnO nanoparticles. As shown in Figure 5, the growth of Skeletocutis odora was inhibited when the concentration of 1, and at concentration of 5, not growth was observed.

Figure 6 shows the growing curves of the fungus in 14 days. We can see the growth rate decreased as the increase of ZnO nanoparticle concentration. And, at concentration 4 and 5, almost no growth in 14 days, indicating the significant inhibition of ZnO.

3.2.3. Species C–Phellinus viticola

The growth of Phellinus viticola was similar to Skeletocutis odora, where the fungus was found sensitive to the presence of ZnO nanoparticles. However, the level of sensitivity was a little bit less than Skeletocutis odora, as we can see a very slow growth of colonies at concentration of 5 (As shown in Figure 7).

Figure 6 shows the growing curves of the fungus in 14 days. We can see the growth rate decreased as the increase of ZnO nanoparticle concentration. However, there was always some growth of fungus colonies at all concentration levels.

3.2.4. Species D–Fomitopsis pinicola

Fomitopsis pinicola is the most special fungus among there four, which found almost not significant inhibition of growth at all concentration level (As shown in Figure 9). All colonies showed almost the same growing speed. As shown in Figure 10, there was only 10% different between the growth at concentration 0 and 5, indicating the resistance of Fomitopsis pinicola to ZnO nanoparticles.

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Figure 3. Photos of Antrodia serialis grown at different concentrations of ZnO nanoparticles and different days (4, 6, 8, 10, 12 days)

Figure 4. The growing curves of Antrodia serialis at different concentration of ZnO nanoparticles.

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Figure 5. Photos of Skeletocutis odora grown at different concentrations of ZnO nanoparticles and different days (4, 6, 8, 10, 12 days).

Figure 6. The growing curves of Skeletocutis odora at different concentration of ZnO nanoparticles.

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Figure 7. Photos of Phellinus viticola grown at different concentrations of ZnO nanoparticles and different days (4, 6, 8, 10, 12 days).

Figure 8. The growing curves of Phellinus viticola at different concentration of ZnO nanoparticles.

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Figure 9. Photos of Fomitopsis pinicola grown at different concentrations of ZnO nanoparticles and different days (4, 6, 8, 10, 12 days).

Figure 10. The growing curves of Fomitopsis pinicola at different concentration of ZnO nanoparticles.

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4. Discussion

4.1. The growth of fungi

Figure 11. Schematic drawing of the size of the fungi after 2, 8 and 14 days growth. Red lines show the size of the fungi without the presence of ZnO, blue line show the size of the fungi in the presence of ZnO.

From the experiments, we can get that the presence of ZnO nanoparticles can actually influence the growth of fungi. However, to different fungi the level of influences are different. For Skeletocutis odora and Phellinus viticola, the presence of nanoparticles obviously influence the growth of the fungi, but for the Antrodia serialis and Fomitopsis pinicola, the presence of nanoparticles has less significant influence. Especially for Fomitopsis pinicola, the mix of nanoparticles almost has no effect on them.

Figure 11 shows the growth rate of four different fungi with and without nanoparticles. In the figure, the left side of vertical axis shows without nanoparticles, the growth situation of different fungi at day 2, day 8, and day 14. The right side of vertical axis shows with nanoparticles at concentration 5, the growth situation of different fungi at day 2, day 8 and day 14. For Antrodia serialis, at the concentration 5, the growth rate is only 60 per cent of without nanoparticles. For Skeletocutis odora, the growth is almost banned by the nanoparticles. In the 14 days, they hardly grew. For Phellinus viticola, the growth rate with nanoparticles is just 10 per cent of without nanoparticles.

Unlike other three species, Fomitopsis pinicola are almost not influenced by the ZnO nanoparticles, the growth rate with nanoparticles is more than 95 percent of without nanoparticles.

Different fungi have different growth phenomenon with nanoparticles from the results we obtained. The reason of this phenomenon is related of the growth characteristics of different species. In the growing process of these species, they will produce some organic acids (From Fredrick’s experimental results), however, the amount of acids they produced are different. The

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total amount of acids that Antrodia serialis and Fomitopsis pinicola produced is more than Skeletocutis odora and Phellinus viticola produced.

These organic acids have significant impacts on the growing process of fungi, especially with ZnO nanoparticles. As we know, that ZnO can have chemical reaction with acid (equation 1), the result of the reaction is the solid ZnO will be dissolved by organic acid, and become the ironic form Zn²⁺. The form changing of zinc makes the nanoparticles will not exist any more, and the characteristics of nanoparticles are gone. However, zinc ions will have influence to the growth now. The extent of fungi growth is depend on two key factors, the first one is how much organic acid the fungi can produce, and the second is whether the fungi is sensitive to the ironic zinc. There should have another factor that is whether do the nanoparticles stimulate the production of organic acids, however, it need more work to discover it.

ZnO + HA → ZnA + H2O (Equation 1)

Figure 12 shows the growth situation with ZnO nanoparticles after 4 days for four different fungi. From the figure, we can easily see there is a transparent circle, which is around the fungi in Fomitopsis pinicola, Antrodia serialis and Phellinus viticola. The circles indicate that the fungi metabolized and produced organic acid, then the acid dissolved the ZnO in the culture media plates. However, the phenomenon did not happen in species Skeletocutis odora.

According to the size of the transparent circle, the order should be like this: Fomitopsis pinicola>Antrodia serialis>Phellinus viticola>Skeletocutis odora. This order represents the amount of acid they produced. Meanwhile, the order is the same as the growth rate with nanoparticles. The results have proved the theory above which is the growth of fungi with nanoparticles depend on the amount acid they produced.

Through further analysis, we can see that there are clearly growth of mycelium in Antrodia serialis and Fomitopsis pinicola. In Phellinus viticola, there is no clear evidence of mycelium growth. This also can prove that the second factor which means the growth of fungi is also related to whether the fungi are sensitive to the ironic zinc. From the results that indicate the Phellinus viticola is the most sensitive species to ironic zinc, then the Antrodia serialis, and the least sensitive one is species Fomitopsis pinicola.

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Figure 12. Photos of fungi after 4 days growth in the presence of ZnO at concentration of 5.

4.2. Eco-toxicity

The four species that we used in our experiment are derived from the Swedish forest. The growth situation of these species are the indicators of the forest, because these situations of these fungi represent the micro eco-system in the soil of the forest, include the pH value of the soil.

We can discuss the influence to the eco-system of ZnO nanoparticles in several levels. First, we assume that the soil environment with single fungus, and then with multiple fungi, in the last we can enlarge into a small forest eco-system

4.2.1. Model 1: The soil environment with single fungus

At first, we start with the simplest model; we assume that in the soil there is only one specie . In this condition, the existence of nanoparticles will infect the soil microenvironment. If only Antrodia serialis or Fomitopsis pinicola exist in the soil, the soil environment will not be changed significantly due to the nanoparticles do not have so much effects on Antrodia serialis or Fomitopsis pinicola. Then, the biological chain will not be changed. The upstream and downstream of the biological chain would be growing normally. Of course, if the concentrates of ironic zinc get higher, it may influence other microorganisms. If it is only Skeletocutis odora in the soil, it will cause the inhibition to the bios, then the biological chain will break at this point, this will cause the big impact on the soil system, and the impact will transmit to the vegetation cover, and even enlarge to the forest eco system. If it is only Phellinus viticola in the soil, the impact will between Antrodia serialis and Skeletocutis odora.

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4.2.2. Model 2: The soil environment with multiple fungi

Figure 13. Schematic drawing the competition of Phellinus viticola and Fomitopsis pinicola without and with the presence of ZnO nanoparticles. The solid lines indicate the size of original planted fungus, and the dash lines show the possible size and shape of these two fungi after two weeks growth.

We can extend the single model to the soil environment with multiple fungi models. When there are two species in the soil at the same time, there exists some competing relationship. In a condition without nanoparticles, the competing relation is in a balancing situation. However, when the nanoparticles join in, the balance will be broken, since the nanoparticles influence the fungus differently. We made a simple model with Phellinus viticola and Fomitopsis pinicola to analysis the impact of nanoparticles. In this model, we put two different fungi Phellinus viticola and Fomitopsis pinicola in a cultural plate, and then observe the growth situation Figure 13 gives a conjecture graph of growing when Phellinus viticola and Fomitopsis pinicola are planted in a plate. In Figure 13 the left side of the figure gives the growing situation of Phellinus viticola and Fomitopsis pinicola without nanoparticles. After two weeks, the growing situation of the fungus may happen as the dotted line shows in the figure. The right side of the figure shows the growing situation with nanoparticles. Because the nanoparticles has the inhibition effect on species Phellinus viticola, so the growing rate should be very slow, but the nanoparticles has not so much effect on Fomitopsis pinicola. Then, the growing speed of Fomitopsis pinicola will be much faster than Phellinus viticola, and as the growing of Fomitopsis pinicola, the advantage will be more and more. In the end, may cause the disappearance of Phellinus viticola.

Figure 14 gives the experiment result of the model. In Figure 14, we can see the Phellinus viticola and Fomitopsis pinicola have obviously growing without nanoparticles, and the growing of Fomitopsis pinicola is faster than Phellinus viticola. However, in the plate with ZnO nanoparticles, Fomitopsis pinicola occupied the absolute dominant position.

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Figure 14. The competition of Phellinus viticola and Fomitopsis pinicola with and without the presence of ZnO.

4.2.3. Model 3: Ecotoxicity in forest

If we enlarge the model of the multiple fungi, take account of the vegetation cover, we can analyze the small-scale forest ecosystem. The presence of the nanoparticles will influence the forest ecosystem in small scale from soil to the vegetation.

Due to the presence of nanoparticles, the balance of fungi are changed, result in the inhibition of some fungi’ growth. Further more, the chemical and physical characteristics of the soil also would also be changed, for instance, the pH value in the soil may change. And the vegetation cover also changes as the soil situation changed.

In Figure 15 the transformation of a simple forest ecosystem is shown. The nanoparticles changed the soil characteristics. The forest will be also influenced. For example, some threes that produced berries and nuts get impact from the system; the quality and quantity of the fruits will change. The change will influence on the birds or other animals, who eat these fruits. And the birds and other animals will leave this area that makes the insects without enemies. The increasing insects may have infected on the forest. The worst case will cause the dead of some trees. Other species of trees will substitute the original ones, then attract other species of birds, which totally changed the ecosystem of this area.

Of course, in reality, that the change of natural environment is much more complicated than we thought, but we could not ignore that the possibilities of nanoparticles may change the ecosystem, because our experiment results have approved that the nanoparticles could change the competing relations between fungi. And we need more research and investigation to discuss and develop the impact to the eco-effects of nanoparticles. As the nano industry coming, we have to face these problems soon.

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Figure 15. Schematic model of the Eco-system change due to the pollution of ZnO nanoparticles.

4.2.4. Further work

Based one the results of our experiment, we can see the potential eco-toxicity of ZnO nanoparticles. However, these results are obtained from a simple experimental system, which should be further extended to more complicated system that closes to real nature environment.

For example, the competence of three or more fungi in the presence of nanoparticles should be investigated. Furthermore, more experiment can be done in green house, where we can grow some grass and fungi, and then water the plants with ZnO nanoparticle contaminated water so see how the plants grow. It seems simple, however, it need long time experiment and analysis.

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5. Conclusions

In this project, we studied the influence of ZnO nanoparticles to the growth of four different fungi at different concentrations. We also investigated how the fungi compete with each other at the sample cultural plates under the influence of ZnO. A potential reason is that due to the difference of the amount of organic acid that produced by the fungus and the sensitivity of fungus to zinc ions, the growth of fungus were influenced by ZnO nanoparticles at different level, where the order of the influence effect is Skeletocutis odora>Phellinus viticola>Antrodia serialis>Fomitopsis pinicola. However, this is just a hypothesis that needs to be proved later.

The results of competing growth indicated that the balance of two fungi could be broken by the ZnO nanoparticles, which enhanced the dominating ability of Fomitopsis pinicola. Generally, although ZnO is considered as safe material, we should take care of it when the size goes down to nano level. However, more studies should be down to fully understand the mechanisms and implications when spread in nature.

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http://en.wikipedia.org/wiki/Fungus Retreived 2014-04-09

[16] Nanotechnolgy research in Finland 2011

http://www.nanobusiness.fi/uploads/nano_research_FINAL_lores.pdf Retreived 2014-04-14

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7. Appendices: Original recorded data from the experiments.

The data shows the diameter of the fungi in unit of cm.

*3C1-2 was polluted after 6 days, thus no data afterwards.

Species A

data Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14

0A1-1 1,3 2 2,7 3,6 4,3 5,2 5,7

0A1-2 1,2 2 2,7 3,5 4,4 5,3 5,9

0A1-3 1,3 1,9 2,7 3,5 4,6 5,3 6

0A2-1 1,2 1,7 2,3 2,9 3,5 4,3 5

0A2-2 1,2 1,8 2,6 3,1 3,9 4,5 5,1

0A2-3 1,2 1,8 2,5 3,1 3,9 4,5 5,1

0A3-1 1,33 2,3 3,3 4 5 5,7 6,5

0A3-2 1,34 2,3 3,1 4,2 5,2 5,7 6,4

0A3-3 1,3 2,3 3,1 4 5,1 5,8 6,5

1A1-1 1,3 2 2,8 3,7 4,5 5,3 6

1A1-2 1,3 2 2,8 3,6 4,2 4,9 5,3

1A1-3 1,3 2,3 3,2 3,9 4 4,7 5,3

1A2-1 1,3 1,9 2,7 3,1 3,9 4,4 5,1

1A2-2 1,23 1,82 2,4 2,9 3,5 4 4,6

1A2-3 1,3 2 2,5 3,1 3,7 4,3 4,9

1A3-1 1,3 2,5 3,3 4,2 5 5,5 6,2

1A3-2 1,3 2,4 3,5 4,3 5,1 5,9 6,6

1A3-3 1,37 2,3 3,3 4,1 4,9 5,7 6,3

2A1-1 1,3 1,95 2,8 3,6 4,5 5,5 6,2

2A1-2 1,3 1,9 2,8 3,5 4,3 5,3 5,9

2A1-3 1,3 1,9 2,4 3,2 4,1 5,2 5,5

2A2-1 1,2 1,8 2,5 3 3,1 4,4 4,9

2A2-2 1,2 1,7 2,2 2,8 3,5 4,3 4,8

2A2-3 1,25 1,8 2,5 3 3,8 4,4 5,1

2A3-1 1,5 2,2 3,2 4 5,1 6,1 7,1

2A3-2 1,47 2,4 3,4 4 5 5,8 6,4

2A3-3 1,35 2,3 3,3 4 5,3 5,9 6,6

3A1-1 1,2 1,9 2,8 3,5 4 5,2 5,8

3A1-2 1,3 2 2,7 3,5 4,3 5,4 5,8

3A1-3 1,2 1,9 2,5 3,3 3,9 4,8 5,3

3A2-1 1,3 1,7 2,2 2,6 3,5 4,1 4,7

3A2-2 1,3 1,9 2,5 3 4 4,5 5

3A2-3 1,2 1,6 2,1 2,5 3,8 4,1 4,7

3A3-1 1,42 2,1 2,5 3,1 3,7 4,2 4,5

3A3-2 1,4 1,9 2,1 2,6 3,1 3,6 4

3A3-3 1,33 1,8 2 2,5 3,2 3,6 4,2

4A1-1 1,2 1,8 2,4 3,1 3,7 4,3 4,6

4A1-2 1,3 1,83 2,4 3,1 3,9 4,8 5,1

4A1-3 1,22 1,9 2,4 3,2 4,3 5,1 5,5

4A2-1 1,3 1,7 2,3 2,8 3,7 4,3 5

4A2-2 1,3 2 2,5 3,4 4,1 4,8 5,3

4A2-3 1,3 1,6 1,9 2,4 2,8 3,2 4,8

4A3-1 1,23 1,6 1,9 2,2 2,5 3 3,3

4A3-2 1,3 1,5 1,8 2,3 3,1 3,8 4,3

4A3-3 1,3 1,5 1,8 2,2 2,9 3,4 3,8

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5A1-1 1,2 1,72 2,3 3 3,4 4,2 4,3

5A1-2 1,22 1,5 2 2,6 3,5 3,8 4,2

5A1-3 1,2 1,6 2,1 2,5 3 3,3 3,6

5A2-1 1,3 1,9 2,8 3,3 3,6 3,8 4,1

5A2-2 1,3 1,4 1,8 2,3 2,7 3 3,4

5A2-3 1,22 1,5 1,8 2 2,3 2,5 2,8

5A3-1 1,26 1,4 1,5 1,8 2 2,4 2,9

5A3-2 1,3 1,6 1,8 2,1 2,6 3,1 3,6

5A3-3 1,3 1,7 2,1 2,4 2,7 3,1 3,5

0B1-1 1,2 2 2,8 4 4,8 6,3 6,8

0B1-2 1,3 1,8 2,4 3 4,3 5,2 5,9

0B1-3 1,2 1,9 2,5 3,5 4,7 5,7 6,3

0B2-1 1,2 1,8 2,3 2,7 3,1 3,5 4,2

0B2-2 1,2 1,9 2,2 2,6 3 3,6 4,2

0B2-3 1,3 1,8 2,3 2,7 3,2 3,6 3,9

0B3-1 1,2 2,25 3,4 4,9 5,8 6,9 7,5

0B3-2 1,2 2,2 3,6 4,9 6 7 7,7

0B3-3 1,22 2,1 3,4 4,5 5,9 6,8 8

1B1-1 1,3 1,8 2,6 3,4 4,1 4,8 5,3

1B1-2 1,2 1,9 2,9 3,5 4 4,5 4,9

1B1-3 1,2 1,92 2,7 3,5 4,1 4,5 5

1B2-1 1,3 1,8 2,7 3 3,1 3,5 4,4

1B2-2 1,4 2 2,4 2,8 2,9 3,6 4

1B2-3 1,4 1,9 2,3 2,8 3,4 3,7 4,5

1B3-1 1,3 1,93 2,9 3,8 4,7 5,4 5,9

1B3-2 1,3 2 2,9 3,8 4,2 5,4 5,9

1B3-3 1,3 1,9 3 3,8 4,15 5,1 5,5

Species B

2B1-1 1,2 1,6 1,9 2,1 2,3 2,6 2,7

2B1-2 1,2 1,6 2 2,2 2,5 2,7 2,9

2B1-3 1,2 1,6 2 2,3 2,6 2,6 3

2B2-1 1,3 1,42 1,8 1,9 2,1 2,2 2,4

2B2-2 1,3 1,41 1,8 1,9 2,1 2,3 2,4

2B2-3 1,3 1,5 1,7 2 2 2,5 2,8

2B3-1 1,2 1,27 1,5 1,7 1,75 1,9 1,95

2B3-2 1,2 1,3 1,8 2,1 2,4 2,6 2,9

2B3-3 1,2 1,3 1,6 2 2,2 2,3 2,4

3B1-1 1,2 1,3 1,5 1,5 1,6 1,7 1,7

3B1-2 1,2 1,3 1,6 1,6 1,72 1,9 1,9

3B1-3 1,2 1,3 1,6 1,7 1,8 1,8 2

3B2-1 1,3 1,4 1,4 1,4 1,45 1,52 1,53

3B2-2 1,25 1,3 1,4 1,4 1,45 1,58 1,6

3B2-3 1,22 1,3 1,4 1,5 1,6 1,7 1,7

3B3-1 1,27 1,22 1,4 1,6 1,7 2 2,2

3B3-2 1,22 1,3 1,4 1,5 1,7 1,8 1,9

3B3-3 1,21 1,3 1,6 1,6 1,77 1,8 1,9

4B1-1 1,2 1,3 1,25 1,22 1,27 1,25 1,28

4B1-2 1,2 1,3 1,3 1,22 1,3 1,3 1,33

4B1-3 1,2 1,2 1,3 1,22 1,3 1,3 1,3

4B2-1 1,2 1,2 1,23 1,21 1,25 1,25 1,2

4B2-2 1,22 1,27 1,24 1,24 1,3 1,25 1,25

4B2-3 1,25 1,3 1,3 1,2 1,3 1,3 1,3

4B3-1 1,3 1,28 1,3 1,3 1,33 1,4 1,6

4B3-2 1,3 1,2 1,23 1,2 1,3 1,3 1,4

4B3-3 1,2 1,21 1,28 1,3 1,37 1,46 1,5

5B1-1 1,2 1,28 1,3 1,2 1,25 1,23 1,23

5B1-2 1,2 1,22 1,22 1,22 1,25 1,23 1,2

5B1-3 1,3 1,3 1,22 1,22 1,25 1,23 1,22

Table of Contents

Independent degree project - first cycle

Abstract

Acknowledgements