The analysis of gene changes associated with formation of the mycorrhizal symbiosis between orchid and fungi could have broad implications for plant pathogen interactions. Fungi associated with North American terrestrial orchids were once included in the pathogenic genus Rhizoctonia. This suggests that orchids are able to overcome or utilize normally pathogenic pathways to establish symbioses. A differential display technique was employed to analyze gene changes in orchid in response to a fungus. Samples of RNA from roots of Cypripedium parviflorum var. pubescens (CyPP) grown in the presence or absence of a mycorrhizal fungus; Thanatephorus pennatus, were analyzed using AFLP differential display. Forty-four fragments were selected out of 5000 as being differentially expressed, but only 15 sequences were obtained. Most showed homology to ribosomal genes. Two represented genes believed to be regulated by the mycorrhizal interaction: trehalose-6-phosphate synthase/phosphatase (Tps), which showed down-regulation and nucleotide binding protein (NuBP), which showed up-regulation. The Tps partial clone identifies 2100 bp at the 3' end of the gene and encodes a protein of 667 amino acids. The NuBP gene is approximately 1200bp in length and encodes a protein of 352 amino acids. The Tps gene exists in multiple copies with high expression in roots and low expression in rhizomes and leaves. The NuBP gene exists as a single copy and has a low level of expression in rhizomes and leaves. Expression of Tps is induced by sucrose, but reduced by trehalose. Cultivation of CyPP with non-mycorrhizal fungi did not affect expression of Tps or NuBP. Trehalose induced NuBP expression whereas sucrose did not. A second species of mycorrhizal fungi induced expression of NuBP but reduced expression of Tps. Analysis of Tps expression in Arabidopsis was done using promoter:GUS fusions. The Tps promoter:GUS plants revealed that Tps expression is constitutive in roots. Regulation of Tps driven GUS is expressed throughout seedlings. GUS was not detected in leaves of older plants but was detected in anthers and stigmatic surfaces of flowers. Expression of GUS driven by Tps showed a strong wound response and was present in the junction between siliques and pedicels. / Ph. D.

The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields. Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor. Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates. Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline. In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar. These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields. / Ph. D.

As part of the ongoing loss of global biodiversity, amphibian populations are experiencing declines and extinctions. A primary factor in these declines is the skin disease chytridiomycosis, which is caused by the fungus Batrachochytrium dendrobatidis (Bd). Recent research suggests that the amphibian skin microbiota has anti-Bd activity and may be an important factor in host disease resistance. However, little is known about the basic ecology of this host-microbe symbiosis, such as how much variation there is in microbial symbionts among host species and populations, and the nature of symbiont transmission, culturability, and function. My dissertation research addressed these basic questions in microbial ecology, as well as used a novel system to examine the long-standing ecological theory of community structure-function relationships. First, host-specificity, population-level variation and potential environmental transmission of the microbiota were examined by conducting a field survey of bacterial communities from bullfrogs, newts, pond water, and pond substrate at a single pond, and newts from multiple ponds. There was variation among amphibian host species and populations in their skin symbionts, and, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment. Second, the culturability of amphibian skin bacteria was assessed by directly comparing culture-dependent and -independent bacterial sequences from the same individuals. Although less than 7% of the amphibian skin microbes were captured using R2A medium, most of the dominant bacteria were represented in our cultures, and similar patterns of diversity among four amphibian species were captured with both approaches. Third, the relationship between microbial community structure and function and selective forces shaping structure and function were examined in bullfrogs by tracking microbial community structure and function following experimental manipulation of the skin microbiota and pathogen exposure. Results of this study demonstrated that Bd is a selective force on cutaneous bacterial community structure and function, and suggest that beneficial states of bacterial structure and function may serve to limit infection and negative fitness consequences of Bd exposure. Using a combination of observational and experimental approaches, my dissertation contributes to understanding structure-function relationships of these complex symbiotic communities of vertebrates. / Ph. D.

We live in an era of global change, where emerging infectious diseases such as Ebola, Zika, bird flu, and white nose syndrome are affecting humans, wildlife, and domesticated species at an increasing rate. To understand and predict the dynamic spread of these infectious agents and other symbionts through host populations and communities, we need dynamic mathematical models that accurately portray host-symbiont transmission. But transmission is an inherently difficult process to measure or study, because it is actually a series of interacting processes influenced by abiotic and biotic factors at multiple scales, and thus empirical tests of the transmission function within epidemiological models are rare. Therefore, in this dissertation, I explore factors at the individual, population, and community-levels that influence host contact rates or symbiont transmission success in a common snail-symbiont system, providing a detailed description of the multi-faceted nature of symbiont transmission. From a review of the ecological literature, I found that most models assume that transmission is a linear function of host population density, whereas most empirical studies describe transmission as a nonlinear function of density. I then quantified the net nonlinear transmission-density relationship in a system where ectosymbiotic oligochaetes are directly transmitted among snail hosts, and I explored the ecological mechanisms underlying the nonlinear transmission-density relationship observed in the field via intraspecific transmission success and contact rate experiments in the laboratory. I found that the field results could be explained by heterogeneity in transmission success among snails with different characteristics and nonlinear contact-density relationships caused by non-instantaneous handling times. After I 'unpacked'population-level transmission dynamics into those individual-level mechanistic processes, I used this same approach to examine higher-level ecological organization by describing the mechanistic underpinnings of interspecific or community-level transmission in the same snail-symbiont system. I found that low interspecific transmission rates in the field were the product of opposing interactions between high population densities, high prevalences of infection, and very low interspecific transmission success caused by strong symbiont preferences for their current host species. Unpacking transmission in this way resulted in one of the most detailed empirical studies of transmission dynamics in a wildlife system, and yielded many surprising new insights in symbiont ecology that would not have been discovered with a purely phenomenological or holistic view of transmission. Though simple, linear, and holistic epidemiological models will always be important tools in disease ecology, 'unpacking'transmission rates and adding heterogeneity and nonlinearity to models, as I have done here, will become increasingly important as we work to maximize model prediction accuracy in this era of increased disease emergence. / Ph. D.

Sinorhizobium meliloti (Ensifer meliloti) is a bacterium that will exist saprotrophically in the soil and rhizosphere or as a differentiated bacteroid inside root nodules of the legume genera Medicago, Melilotus, and Trigonella. It exists in symbiosis when inside a host plant and will fix gaseous N2 into ammonium for the plant. In return, a population of the bacteria is harbored inside the plant where it can proliferate beyond what would be possible in the rhizosphere or bulk soil. This symbiosis is a defining feature of the Fabaceae (legume) family, a clade that diverged approximately 60 million years ago and is now the 5th largest plant family by species count. Each legume species pairs with one or several strains of bacteria, referred to broadly as rhizobia. The rhizobia identify their proper host plant by a cocktail of secondary metabolites called flavonoids released from specific parts of the roots. Initiation of the symbiosis may only occur at the tips of young root hairs. Therefore, the means rhizobia take to localize themselves to these sites must be the inceptive step in the symbiotic interaction. The studies here examine the mechanisms and priorities rhizobia use to achieve this goal. Movement of bacteria is referred to as motility and is achieved via (in rhizobia, multiple) rotating flagella, proteinaceous extracellular appendages that propel the cell through liquid environments. On their own, flagella may only move but not guide the cell. Navigation is achieved through sensors that detect chemical attractant or repellent cues in the environment and an intracellular signaling system that relays information to appropriately control locomotion. This sensing is called chemotaxis. A research focus is directed on the sensing aspect of chemotaxis to understand which chemical compounds are the preferred attractants for S. meliloti. An emphasis is placed on those compounds released from germinating host seeds. Chapter 2 spearheads our research goals by examining the chemotactic potential of host-derived flavonoids, the compounds that induce the symbiotic signaling in the rhizobial symbiont. While a logical place to start, this study reveals that our strain of rhizobia is not attracted to flavonoids. We determined that the best chemoattractants are hydrophilic in nature and that hydrophobic compounds, such as flavonoids, are not effective chemoattractants. In addition, we discuss the nature of chemotactic agents and symbiosis inducers to fortify our understanding of how classes of compounds contribute to the rhizobia-plant interaction. In chapter 3, we characterize the sensor protein, McpV, and its ligand profile for carboxylates. The protein is first screened using a high-throughput assay to test numerous possible ligands simultaneously. We confirm positive reactions using direct binding studies and quantify dissociation constants. Then, the phenotypic response to these ligands is measured using capillary chemotaxis assays, and the role mcpV plays in this response is confirmed using deletion mutants. Last, the symbiotic context is addressed by quantifying these ligands in exudates of the host alfalfa. These experiments show that McpV is a chemotactic sensor dedicated to detecting 2 – 4 C monocarboxylates. Only one of the compounds found in the ligand profile, glycolate, was detected in seed exudates, so the contribution of McpV to host sensing is yet to be expounded. Chapter 4 follows the model of chapter 2 but is complicated when the ligand screen used previously gives ambiguous results. Using direct binding studies, we were able to confirm the true ligand amidst numerous false positives. Analytical gel filtration suggests that McpT exists as a dimer regardless of ligand binding. Capillary chemotaxis assays quantified the responses mediated by McpT to di- and tri-carboxylates, which were slightly weaker, but still on-par with the responses to McpV ligands. Strains with mcpT deletions showed strongly reduced, but in some cases, not abolished, chemotaxis to carboxylates. Chapter 5 examines McpX – the chemoreceptor already known to be a sensor of quaternary ammonium compounds. This is a structural investigation into the binding of McpX to its ligands. A crystal structure of the ligand binding region of the protein is resolved to understand how ligands fit into the binding pocket of McpX and what determines its structurally diverse ligand profile. The contribution of certain residues to ligand binding are further probed using direct binding studies on single point variants of McpX. The analysis of chemoreceptor functions hint at what kinds of molecules are most important to bacterial survival and reproduction. Knowing what the bacterium is tuned to seek out grants understanding of what niches they prefer, and how they thrive in those niches. For S. meliloti and other rhizobia, the preeminent niche is one in symbiosis with a host plant. The sum of this knowledge we have accrued with S. meliloti lends itself to agricultural goals of soil enrichment, legume inoculation, nutrient cycling, and environmentally safe and efficient crop fertilization. / Doctor of Philosophy / Sinorhizobium meliloti and other soil-dwelling bacteria termed rhizobia are crucial to the cultivation of leguminous crops such as alfalfa, soy, pea, lentil, peanut, and many more. The bacterium can be internalized by the plant host's roots where it will supply the plant with nitrogen. This is a great boon to crops when they need to accumulate more protein in seed stores, or for plants that survive in nutrient depleted soils. The bacterium must begin seeking out the host plant by sensing chemical cues. It can navigate to the proper location by using a process called chemotaxis. This process is centered around chemoreceptors that can be likened to the nose of the bacterium. Using these chemoreceptors, the bacterium will seek out compounds that benefits it – these are usually food sources. Identifying what each individual chemoreceptor senses allows us to understand what the bacterium needs to seek out to survive. We correlate this information with compounds that the plant secretes and find that many chemoreceptors have evolved to sense signals that will lead the bacterium to a plant root. This interaction is a key part of how the symbiosis is propagated and ultimately benefits the agriculture of leguminous plants.

Since available phosphate (Pi) resources in soil are limited, symbiotic interactions between plant roots and arbuscular mycorrhizal (AM) fungi are a widespread strategy to improve plant phosphate nutrition. The repression of AM symbiosis by a high plant Pi-status indicates a link between Pi homeostasis signalling and AM symbiosis development. This assumption is supported by the systemic induction of several microRNA399 (miR399) primary transcripts in shoots and a simultaneous accumulation of mature miR399 in roots of mycorrhizal plants. However, the physiological role of this miR399 expression pattern is still elusive and offers the question whether other miRNAs are also involved in AM symbiosis. Therefore, a deep sequencing approach was applied to investigate miRNA-mediated posttranscriptional gene regulation in M. truncatula mycorrhizal roots. Degradome analysis revealed that 185 transcripts were cleaved by miRNAs, of which the majority encoded transcription factors and disease resistance genes, suggesting a tight control of transcriptional reprogramming and a downregulation of defence responses by several miRNAs in mycorrhizal roots. Interestingly, 45 of the miRNA-cleaved transcripts showed a significant differentially regulated between mycorrhizal and non-mycorrhizal roots. In addition, key components of the Pi homeostasis signalling pathway were analyzed concerning their expression during AM symbiosis development. MtPhr1 overexpression and time course expression data suggested a strong interrelation between the components of the PHR1-miR399-PHO2 signalling pathway and AM symbiosis, predominantly during later stages of symbiosis. In situ hybridizations confirmed accumulation of mature miR399 in the phloem and in arbuscule-containing cortex cells of mycorrhizal roots. Moreover, a novel target of the miR399 family, named as MtPt8, was identified by the above mentioned degradome analysis. MtPt8 encodes a Pi-transporter exclusively transcribed in mycorrhizal roots and its promoter activity was restricted to arbuscule-containing cells. At a low Pi-status, MtPt8 transcript abundance inversely correlated with a mature miR399 expression pattern. Increased MtPt8 transcript levels were accompanied by elevated symbiotic Pi-uptake efficiency, indicating its impact on balancing plant and fungal Pi-acquisition. In conclusion, this study provides evidence for a direct link of the regulatory mechanisms of plant Pi-homeostasis and AM symbiosis at a cell-specific level. The results of this study, especially the interaction of miR399 and MtPt8 provide a fundamental step for future studies of plant-microbe-interactions with regard to agricultural and ecological aspects. / Phosphat ist ein essentieller Bestandteil der pflanzlichen Ernährung und ein Mangel führt zu schwerwiegenden Folgen für Wachstum, Entwicklung und Reproduktion der Pflanze. Eine der wichtigsten Strategien, um einen Mangel an löslichem Phosphat im Boden auszugleichen, ist die arbuskuläre Mykorrhiza, einer Wurzelsymbiose zwischen Pflanzen und im Boden lebenden Mykorrhizapilzen. Die Symbiose dient dem gegenseitigen Nährstoffaustausch, der über bäumchenartige Strukturen in Wurzelzellen, den Arbuskeln, realisiert wird. Über ein weit reichendes Netzwerk im Boden verbessert der Pilz die Phosphatversorgung der Pflanzen, wohingegen die Pflanze photosynthetisch erzeugte Zucker zur Verfügung stellt. Ein erhöhter Phosphatgehalt in der Pflanze führt zur Unterdrückung der Symbiose. Da weitestgehend unbekannt ist, wie genau Pflanzen diese Einschränkung der Symbiose regulieren, kann die Erforschung dieses Zusammenhangs einen wichtigen Beitrag für Agrarwirtschaft und Umweltschutz leisten. Im Rahmen dieser Arbeit konnte durch die Entdeckung eines neuen, bisher unbekannten Zielgens aufgezeigt werden, dass die für den Ausgleich des pflanzlichen Phosphathaushalts wichtige Mikro-RNA (miR) 399 auch in der Regulation der arbuskulären Mykorrhizasymbiose von besonderer Bedeutung ist. MiRNAs regulieren die Aktivität von Zielgenen indem sie die jeweiligen Transkripte durch Bindung für den Abbau markieren. In kolonisierten Wurzeln, insbesondere in arbuskelhaltigen Wurzelzellen, konnte eine erhöhte Anhäufung der miR399 beobachtet werden. Durch das Verfahren der Hochdurchsatz-Sequenzierung des Wurzeldegradoms, bei dem alle abgebauten Transkripte analysiert werden, konnte das neue Zielgen der miR399 Familie, MtPT8, identifiziert werden. Dieses codiert für einen Phosphat-Transporter, der diesen Studien zufolge ausschließlich in mykorrhizierten Wurzeln vorkommt und dessen Transkription auf arbuskelhaltige Zellen beschränkt ist. Mit der Identifizierung dieses neuen Zielgens konnte erstmals der Beweis für die direkte Verbindung der pflanzlichen Phosphathomöostase durch miR399 und der arbuskulären Mykorrhizasymbiose gezeigt werden. Die Untersuchung der physiologischen Funktion dieses mykorrhizaspezifischen Phosphat-Transporters bietet die Möglichkeit, die Zusammenhänge der phosphatabhängigen Regulation der Symbiose aufzuklären und weit reichende Einblicke in die Regulationsmechanismen während der Pflanze-Pilz-Interaktion zu erhalten.

As the world's population increases, so does the use of resources and thus also the amount of waste. This is an unsustainable resource use trend, with low resource efficiency and high throughput, but can be counteracted with a circular economy. In recent years, an increasing number of initiatives have been taken to strengthen sustainability work. On a global level, the UN developed Agenda 2030, and in 2020 the Swedish government presented a strategy for the transition to a circular economy in Sweden. One way to contribute to increased sustainability and a transition to a circular economy is through industrial and urban symbiosis (IUS). IUS is about increasing resource utilization through greater collaboration between organizations. One organization's residuals and waste can be another's resource. In addition to physical flows, IUS can involve the exchange of information and knowledge between different actors, or the sharing of services or equipment. Urban symbiosis means that the needs, flows and relationships of the city or urban areas are combined with those of industries. Waste contractors, universities, government agencies and production companies can all contribute to this collaboration. For IUS to develop in a planned way, a coordinating or facilitating actor is needed who can work both comprehensively and impartially. This role could be carried out by a municipality and according to the Swedish Instrument of Government, municipalities have a responsibility to try to act towards sustainability. In addition, many municipalities have directly or indirectly linked their strategies and overall goals to the sub-goals of Agenda 2030. Thus, there are many potential benefits for municipalities in achieving their sustainability objectives by implementing IUS in their operations. Since the 2000s, more and more research has been carried out on IUS, and an increasing number of municipalities have begun implementing it. However, most research focuses on private actors, and does not address the unique opportunities and limitations that a municipal organization may have, considering they are a politically controlled organization. There is a lack of an overall mapping, compilation, and tools for what specifically Swedish municipalities can have and use, in the form of drivers, barriers, approaches and consequences, when working with IUS. The purpose of this study has thus been to map how municipalities, within their existing organization, can include and enable the development of industrial and urban symbiosis, and create a tool to facilitate that development. To answer the research questions and fulfil the purpose, a qualitative case study was conducted through a literature review and a semi-structured interview study of five municipalities that have already initiated the development of IUS in their organizations. The five municipalities have been Sotenäs, Malmö, Helsingborg, Lidköping and Gotland and the selected respondents were municipal employees and elected politicians from four areas of the municipal organization. 19 individual interviews were recorded and transcribed, and the responses were then categorized as a driver, barrier, process, or consequence of working with IUS. The categorization of the collected empirical data was compiled into five tables which showed that 30 drivers, 32 barriers, 56 approaches, 32 positive consequences and 6 negative consequences had been mentioned during the interviews. The most prominent drivers were that the municipal organization is development-driven, there is strong political support early on, there is a large network of contacts and cooperation within the region, as well as the presence of champions. Several prominent obstacles were highlighted and the most obvious of these were the need for and the time required to create political anchoring, the organizational culture, and the lack of knowledge that comes with the fact that IUS is a complex subject that can be difficult to explain. The interview study also resulted in many approaches, which differed between the municipalities. However, there were some specific ones that all municipalities emphasized as important for the development of IUS. Some examples of these were collaborating with scientific academics, working more cross-sectorally, and taking inspiration from other municipalities. When it comes to consequences, most municipalities that have come quite far with the work have experienced at least one negative consequence, but the positive consequences are significantly more. Based on these tables, a tool was developed: Matrisen (‘The Matrix’), which is divided into three parts; 1. Identify the municipality's current situation based on factors, 2. Examine which processes can be used to influence the current situation, and 3. Make an action plan. The Matrix is designed as a focus group interview with inspiration from a workshop, which is intended to be part of a larger workshop that representatives from a municipality will participate in to begin its work to enable IUS in its municipality. The Matrix is also intended to be an iterative process that will be used continuously over the years. When the content of the tool was compared to scientific literature, several connections for most factors and processes were found. Unfortunately, the tool could not be tested and thus there are several risks and opportunities for improvement, but it is still considered to be of great value as it is unique in that it is the first tool focused on simplifying work with IUS for specifically Swedish municipalities. Another thing that makes the Matrix unique is that it both gives an overall view and is generalizing, while it at the same time provides the opportunity to be individual. It also focuses on developing the fundamental aspects needed for a municipality to be able to initiate work with IUS. There are many reasons why municipalities should work to implement IUS. Municipalities can, for example, contribute with a neutral meeting place because they do not have to act profitably or are exposed to rivalry in the same way as companies. In addition, the municipalities are responsible for some of society’s large systems with important flows and have a public responsibility by having to handle certain constitutional tasks. This means that the municipalities will be part of an IUS even if they do not actively choose to get involved. For these reasons, they can benefit others by also acting on several of the relevant roles needed in an IUS. For their own benefit, municipalities should work with IUS to help them achieve their business and sustainability goals quicker. Working with IUS, for a municipal organization, also brings benefits for other actors within the municipality and can affect the municipality's reputation in a positive way. Swedish municipalities should therefore work to introduce IUS in their organization, based on their own circumstances and capacity. / I takt med att jordens befolkning ökar, ökar också resursanvändningen och därmed även avfallsmängderna. Detta är en ohållbar resursanvändningstrend eftersom det innebär låg resurseffektivitet och hög genomströmningshastighet, vilket är något som cirkulär ekonomi motverkar. Under de senaste åren har ett ökande antal initiativ tagits för att stärka hållbarhetsarbetet; på global nivå har FN skapat Agenda 2030 och 2020 presenterade den svenska regeringen en strategi för omställningen till cirkulär ekonomi i Sverige. Ett sätt att bidra till ökad hållbarhet och cirkulär ekonomi är genom industriell och urban symbios (IUS). IUS handlar om att försöka öka resursutnyttjandet genom större samverkan mellan organisationer. En organisations restprodukt och avfall kan bli en annans resurs och utöver fysiska flöden kan det även handla om att öka utbytet av information och kunskap mellan olika aktörer eller att flera delar tjänster eller utrustning med varandra. Urban symbios innebär att stadens eller urbana områdens behov, flöden och relationer kombineras med de hos industrier. Så väl avfallsentreprenörer, universitet, myndigheter och producerande företag kan medverka till denna samverkan. För att IUS ska kunna utvecklas på ett planerat sätt behövs någon form av koordinerande eller faciliterande aktör som kan arbeta på ett övergripande och opartiskt sätt. Den koordinerande aktören skulle förslagsvis kunna vara en kommun och enligt Regeringsformen har kommuner ett ansvar i att försöka agera för hållbarhet. Flertalet kommuner har direkt eller indirekt kopplat sina strategier och övergripande mål till delmålen i Agenda 2030. Därmed finns det många potentiella vinster för en kommun och dess hållbarhetsarbete med att initiera IUS i sin verksamhet. Det har under 2000-talet börjat genomföras alltmer forskning inom ämnet IUS och allt fler kommuner har börjat arbeta med det. Den mesta forskningen fokuserar dock på allmänna aktörer, och tar inte in de unika möjligheter och begränsningar som en kommunorganisation kan ha, i och med att de är en politiskt styrd organisation. Det saknas en övergripande kartläggning, sammanställning och verktyg för vad specifikt svenska kommuner kan ha för drivkrafter, hinder, tillvägagångssätt och följder av att arbeta med IUS. Syftet med denna studie har därmed varit att kartlägga hur kommuner, inom sin befintliga organisation, kan inkludera och möjliggöra utvecklingen av industriell och urban symbios, samt skapa ett verktyg för att underlätta den utvecklingen. För att svara på frågeställningarna och uppfylla syftet genomfördes en kvalitativ fallstudie genom en litteraturgranskning samt en semistrukturerad intervjustudie av fem kommuner som redan har initierat utvecklingen av IUS i sina organisationer. De fem kommunerna har varit Sotenäs, Malmö, Helsingborg, Lidköping och Gotland och de utvalda respondenterna var tjänstepersoner och förtroendevalda politiker från fyra områden av kommunorganisationen. 19 individuella intervjuer spelades in och transkriberades där svaren sedan kategoriserades som en drivkraft, hinder, process eller följd av arbete med IUS. Kategoriseringen av den insamlade empirin samanställdes i fem tabeller som visade att 30 drivkrafter, 32 hinder, 56 tillvägagångssätt, 32 positiva följder samt 6 negativa följder hade nämnts under intervjuerna. De mest framträdande drivkrafterna var att kommunorganisationen är utvecklingsdriven, att det finns en stark politisk stöttning tidigt, att det finns ett stort kontaktnät samt samarbete inom regionen och även förekomsten av eldsjälar. Det uppmärksammades flertalet framträdande hinder och de tydligaste av dessa var behovet av och tidsåtgången som krävs för att skapa politisk förankring, organisationskulturen samt kunskapsbristen som kommer med att IUS är ett komplext ämne som kan vara svårt att förklara. Intervjustudien resulterade även i ett stort antal tillvägagångssätt som skiljde sig kommunerna mellan, men det fanns vissa specifika som samtliga framhävde som viktiga för utvecklingen av IUS. Några exempel på dessa var att samarbeta med vetenskaplig akademi, arbeta mer tvärsektoriellt, samt ta inspiration från andraVkommuner. När det kommer till följder har de flesta kommuner som kommit en bit med arbetet upplevt någon enstaka negativ följd, men de positiva följderna är betydligt fler. Utifrån dessa tabeller togs ett verktyg fram: Matrisen, som är uppdelad i tre delar; 1. Identifiera kommunens nuläge utifrån faktorer, 2. Undersöka vilka processer som kan användas för att påverka nuläget, samt 3. Göra en handlingsplan. Matrisen är utformad som en fokusgruppsintervju med inspiration av workshop som är tänkt att vara en del av en större workshop som representanter från en kommun ska delta i för att påbörja sitt arbete med att möjliggöra IUS i sin kommun. Matrisen är också avsedd att var en iterativ process som ska användas kontinuerligt genom åren. När verktygets innehåll jämfördes mot vetenskaplig litteratur återfanns flertalet kopplingar för de flesta faktorer och processer. Verktyget kunde dessvärre inte testas och därmed finns det ett antal risker och förbättringsmöjligheter, men den anses ändå vara av stort värde då den är unik i form av att det är det första verktyget som är inriktat på att förenkla arbetet med IUS specifikt för svenska kommuner. Något annat som gör Matrisen unik är att den är både översiktlig och generaliserande, samtidigt som den ger möjlighet att vara individuell. Den fokuserar också på att ta fram de fundamentala aspekterna som behövs för att en kommun ska kunna initiera arbete med IUS. Det finns många anledningar på varför kommuner bör arbeta med att möjliggöra IUS. Kommuner kan exempelvis bidra med en neutral mötesplats eftersom de inte ska agera vinstdrivande eller är konkurrensutsatta på samma sätt som företag. Dessutom har kommunerna ansvar för stora samhällsystem med viktiga flöden samt har ett samhällsansvar genom att de ska sköta vissa lagstadgade uppgifter. Det leder till att kommunerna kommer vara en del av en IUS även om de inte aktivt väljer att engagera sig. Av dessa anledningar kan de göra nytta för andra genom att också agera flera av de relevanta rollerna som behövs i en IUS. För sin egen skull bör kommuner arbeta med IUS för att det kan hjälpa dem att snabbare nå sina uppsatta strategier och mål för exempelvis näringsliv och hållbarhet. Arbete med IUS för en kommunorganisation medför även vinster för övriga verksamheter i kommunen samt kan påverka kommunens rykte på ett positivt sätt. Svenska kommuner bör därför arbeta för att införa IUS i sin organisation, utifrån sina egna förutsättningar och kapacitet. / Cirkulär Ekonomi Genom Industriell Samverkan 2.0

Os Parques Eco-Industriais (EIP - Eco-Industrial Park) surgiram como um novo modelo de organização espacial para arranjos industriais. Uma característica importante para um EIP é a adoção do conceito de simbiose industrial (SI), em que as empresas reutilizam resíduos em busca de um sistema fechado, diminuindo impacto ambiental. Há propostas para avaliar o fluxo de resíduos e a simbiose de um EIP por meio de indicadores detalhados em um determinado momento. Porém, trata-se de uma visão estática do parque na qual seria possível que os agentes implementassem mudanças para construir uma situação de sistema fechado ideal. Pesquisas recentes demonstram que essa abordagem enfrenta algumas limitações, uma vez que o processo de simbiose depende das decisões rotineiras e individuais de cada ator, empresários, técnicos e agenciadores, as quais podem alterar a dinâmica do fluxo de material no decorrer do tempo. A fim de contribuir, propõe-se um indicador de simbiose industrial diferente, elaborado segundo a abordagem do parque como um sistema dinâmico. Trata-se de uma ferramenta capaz de monitorar o nível de simbiose de um parque ao longo do tempo. Diferencia-se por permitir que a organização agenciadora possa implementar políticas de incentivo à simbiose industrial, influenciando nas decisões rotineiras dos diversos agentes do parque, em prol da simbiose. Empregaram-se estudos de caso, revisão sistemática da literatura e análise teórico-conceitual. Os primeiros resultados foram dois estudos de casos em parques que se autodenominavam EIPs, identificando oportunidades para a promoção da simbiose. O segundo resultado foi a proposta teórico-conceitual do Indicador de Simbiose Industrial (ISI) que pode ser utilizado como instrumento para o ambiente industrial dinâmico. Por fim, apresenta-se a análise das propriedades do indicador a partir de dados obtidos em um dos casos. Os resultados indicaram que o ISI pode identificar níveis de simbiose distintos para um mesmo parque, conforme sua evolução em direção a uma simbiose perfeita. A avaliação da coerência do ISI permitiu demonstrar sua capacidade em captar a ausência e a presença do processo de simbiose, atendendo as expectativas iniciais da construção do indicador. A avaliação do comportamento do ISI por meio da variação dos seus elementos permitiu analisar o indicador sob diferentes possibilidades, correspondendo positivamente às expectativas da pesquisa. Indica-se também várias questões que precisam ser verificadas para a validação do indicador e os próximos passos para torná-lo uma ferramenta útil para o gerenciamento de parques industriais interessados em aprimorar o seu nível de simbiose industrial. / The Eco-Industrial Parks (EIP - Eco-Industrial Park) emerged as a new model of spatial organization for industrial arrangements. An important feature for an EIP is the adoption of the concept of industrial symbiosis (IS), in which companies reuse waste to reach a closed system, reducing environmental impact. There are proposals to assess the waste stream and the symbiosis of an EIP through detailed indicators at a determined time. However, it is a static view of the park in which would be possible to the managers implement changes to construct a state of ideal closed system. Recent researches show that this approach faces some limitations, once the process symbiosis depends on the routine and individual decisions of the actors, managers, technicians and agents, which can alter the dynamics of the material flow through time. In order to contribute, it is proposed an different indicator of industrial symbiosis, prepared according to the approach of the park as a dynamic system. This is a tool that can monitor the level of symbiosis of a park during a determined time. This is different because allow the agent organization to implement policies to encourage industrial symbiosis influencing the routines decisions of the parks agents in favor of symbiosis. It was made case studies, systematic review of the literature and theoretical and conceptual analysis. The first results were two case studies in parks that called themselves EIPs, identifying opportunities to promote symbiosis. The second result was the proposed theoretical-conceptual Indicator Industrial Symbiosis (ISI) which can be used as a tool for the dynamic industrial environment. Finally, it shows the analysis of the indicator properties considering data obtained in one of the cases. The results indicated that the ISI can identify different levels of symbiosis for the same park as its evolution toward a perfect symbiosis. The assessment of the consistency of the ISI has demonstrated its ability to capture the absence and presence of the symbiosis process, confirming the expectations of the initial construction of the indicator. The performance evaluation of the ISI through the variation of its elements, allowed us to analyze the indicator under different possibilities, corresponding positively to the expectations of the research. Also, it indicates that several issues need to be checked to validate the indicator and the next steps to make it a useful tool for managing industrial parks interested in improving their level of industrial symbiosis.

Os Parques Eco-Industriais (EIP - Eco-Industrial Park) surgiram como um novo modelo de organização espacial para arranjos industriais. Uma característica importante para um EIP é a adoção do conceito de simbiose industrial (SI), em que as empresas reutilizam resíduos em busca de um sistema fechado, diminuindo impacto ambiental. Há propostas para avaliar o fluxo de resíduos e a simbiose de um EIP por meio de indicadores detalhados em um determinado momento. Porém, trata-se de uma visão estática do parque na qual seria possível que os agentes implementassem mudanças para construir uma situação de sistema fechado ideal. Pesquisas recentes demonstram que essa abordagem enfrenta algumas limitações, uma vez que o processo de simbiose depende das decisões rotineiras e individuais de cada ator, empresários, técnicos e agenciadores, as quais podem alterar a dinâmica do fluxo de material no decorrer do tempo. A fim de contribuir, propõe-se um indicador de simbiose industrial diferente, elaborado segundo a abordagem do parque como um sistema dinâmico. Trata-se de uma ferramenta capaz de monitorar o nível de simbiose de um parque ao longo do tempo. Diferencia-se por permitir que a organização agenciadora possa implementar políticas de incentivo à simbiose industrial, influenciando nas decisões rotineiras dos diversos agentes do parque, em prol da simbiose. Empregaram-se estudos de caso, revisão sistemática da literatura e análise teórico-conceitual. Os primeiros resultados foram dois estudos de casos em parques que se autodenominavam EIPs, identificando oportunidades para a promoção da simbiose. O segundo resultado foi a proposta teórico-conceitual do Indicador de Simbiose Industrial (ISI) que pode ser utilizado como instrumento para o ambiente industrial dinâmico. Por fim, apresenta-se a análise das propriedades do indicador a partir de dados obtidos em um dos casos. Os resultados indicaram que o ISI pode identificar níveis de simbiose distintos para um mesmo parque, conforme sua evolução em direção a uma simbiose perfeita. A avaliação da coerência do ISI permitiu demonstrar sua capacidade em captar a ausência e a presença do processo de simbiose, atendendo as expectativas iniciais da construção do indicador. A avaliação do comportamento do ISI por meio da variação dos seus elementos permitiu analisar o indicador sob diferentes possibilidades, correspondendo positivamente às expectativas da pesquisa. Indica-se também várias questões que precisam ser verificadas para a validação do indicador e os próximos passos para torná-lo uma ferramenta útil para o gerenciamento de parques industriais interessados em aprimorar o seu nível de simbiose industrial. / The Eco-Industrial Parks (EIP - Eco-Industrial Park) emerged as a new model of spatial organization for industrial arrangements. An important feature for an EIP is the adoption of the concept of industrial symbiosis (IS), in which companies reuse waste to reach a closed system, reducing environmental impact. There are proposals to assess the waste stream and the symbiosis of an EIP through detailed indicators at a determined time. However, it is a static view of the park in which would be possible to the managers implement changes to construct a state of ideal closed system. Recent researches show that this approach faces some limitations, once the process symbiosis depends on the routine and individual decisions of the actors, managers, technicians and agents, which can alter the dynamics of the material flow through time. In order to contribute, it is proposed an different indicator of industrial symbiosis, prepared according to the approach of the park as a dynamic system. This is a tool that can monitor the level of symbiosis of a park during a determined time. This is different because allow the agent organization to implement policies to encourage industrial symbiosis influencing the routines decisions of the parks agents in favor of symbiosis. It was made case studies, systematic review of the literature and theoretical and conceptual analysis. The first results were two case studies in parks that called themselves EIPs, identifying opportunities to promote symbiosis. The second result was the proposed theoretical-conceptual Indicator Industrial Symbiosis (ISI) which can be used as a tool for the dynamic industrial environment. Finally, it shows the analysis of the indicator properties considering data obtained in one of the cases. The results indicated that the ISI can identify different levels of symbiosis for the same park as its evolution toward a perfect symbiosis. The assessment of the consistency of the ISI has demonstrated its ability to capture the absence and presence of the symbiosis process, confirming the expectations of the initial construction of the indicator. The performance evaluation of the ISI through the variation of its elements, allowed us to analyze the indicator under different possibilities, corresponding positively to the expectations of the research. Also, it indicates that several issues need to be checked to validate the indicator and the next steps to make it a useful tool for managing industrial parks interested in improving their level of industrial symbiosis.

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