Harvesting history affects soil respiration and litterfall but not overall carbon balance in boreal Norway spruce forests

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Et sagn om lys og råte : molekylære responser til UV-C lys i jordbær og gråskimmelsopp

The desire for sustainable agricultural practices requires innovative approaches for disease management and crop quality enhancement. Ultraviolet-C (UV-C) light has emerged as a promising scalable alternative to agrochemicals to control phytopathogenic fungi with minimal environmental impact. The high photon energy in UV-C wavelength (254 nm) can inactivate a wide range of microorganisms by disrupting their DNA and RNA. Delivery of low doses of UV-C minimises the damage to plants but may fall short of delivering the lethal dose needed for more tolerant pathogenic fungi. Botrytis cinerea, a primary cause of grey mould in strawberries (Fragaria × ananassa), can significantly reduce crop yield, quality and postharvest storage. The work presented in this thesis explores the molecular and physiological responses of B. cinerea and F. × ananassa to UV-C treatment, aiming to elucidate how it can operate as a dual-function tool for controlling pathogens and enhancing strawberry plant defences. The first study of this thesis focused on how B. cinerea responded to varying doses of UV-C in vitro. It revealed that a low-dose UV-C (5 mJ/cm²) delayed conidial germination and differentially regulated genes associated with melanin biosynthesis. Contrary to expectations, melanin concentration decreased during early fungal development despite UV-C treatment, suggesting that B. cinerea does not rely on melanin for UV-C-induced protection. Building on this, the subsequent studies explored the more complex in vivo interaction by using strawberry fruits treated with regular, commercial-level preharvest UV-C applications, followed by infection with B. cinerea. After a low-dose postharvest UV-C treatment, transcriptomic analysis of strawberry fruit revealed downregulation of key genes in the phenylpropanoid biosynthetic pathway, while genes encoding flavonoid transporters and various peroxidases were upregulated. These results suggested that flavonoid transport and enhanced oxidative stress responses are key aspects of the strawberry response to combined stress factors. Yet, the final study revealed an increase in flavonol glycoside concentrations in response to postharvest UV-C treatment. Both preharvest and postharvest UV-C applications led to significant increases in kaempferol 3-coumaroylglucoside and quercetin 3-glucuronide, important compounds for plant photoprotection, oxidative stress response, and defence capacity. Taken together, these responses highlighted the ability of UV-C treatment to simultaneously control fungal pathogens and augment plant resilience through phytonutrient accumulation. The thesis discussion centres on the temporal dynamics of these responses, highlighting how UV-C treatment elicited transient fluctuations in both pathogen and host. By dissecting these dynamics, the thesis provides insight into how the timing of the UV-C application and developmental stage of the organism influenced gene expression, metabolite production, and phenotypic outcomes. For example, UV-C treatments of B. cinerea conidia delayed germination and modulated melanin biosynthetic genes, while treatments during infection on strawberry fruit revealed nuanced changes in secondary metabolite pathways, such as those related to flavonoid biosynthesis. This work also discusses the potential for preharvest UV-C treatment to act as a priming agent, preparing the plant for enhanced defensive responses. While UV-C during the growing season was not explicitly optimised for priming, observations of increased flavonoid accumulation and transcriptomic shifts suggest that sub-lethal UV-C exposure may enhance resilience against pathogens. Such effects could be considered inadvertent yet beneficial byproducts of UV-C application, warranting further investigation into priming mechanisms. The discussion synthesises these findings to propose how UV-C treatment can be strategically integrated into agricultural practices, balancing immediate pathogen control with potential long-term benefits to plant defence.Ønsket om en bærekraftig landbrukspraksis krever innovative tilnærminger til sykdomsbekjempelse og kvalitetsforbedring av avlinger. Ultrafiolett-C (UV-C)-lys fremstår som et lovende og bærekraftig alternativ til landbrukskjemikalier for bekjempelse av fytopatogene sopper, samtidig som miljøpåvirkningen holdes minimal. Bruk av lave doser UV-C minimerer imidlertid skadene på plantene, men kan være utilstrekkelig for å gi den dødelige dosen som er nødvendig for mer tolerante patogene sopper. Botrytis cinerea som er den viktigste årsaken til gråskimmel i jordbær (Fragaria × ananassa), kan redusere avling, kvalitet og lagring etter høsting betydelig. Arbeidet som presenteres i denne avhandlingen, utforsker derfor de molekylære og fysiologiske responsene til B. cinerea og F. × ananassa ved UV-C-behandling, med sikte på å belyse UV-C som et verktøy med to funksjoner for å bekjempe patogener og forbedre jordbærplantenes forsvarsevne. Den første studien i denne avhandlingen fokuserte på B. cinerea in vitro, der ulike doser UV-C ble brukt. Det viste seg at lavdose UV-C (5 mJ/cm²) forsinket spiring av konidier og differensialregulerte gener forbundet med melaninbiosyntese. I motsetning til hva som var forventet, sank melaninkonsentrasjonen under tidlig sopputvikling til tross for UV-C-behandling. Dette kan tyde på at B. cinerea ikke er avhengig av melanin for UV-C-indusert beskyttelse. På bakgrunn av dette utforsket de påfølgende studiene en mer kompleks in vivo-interaksjon ved å bruke jordbærfrukter som ble behandlet med vanlig, kommersiell UV-C-behandling før høsting, etterfulgt av infeksjon med B. cinerea. Etter en lavdose UV-C-behandling etter innhøsting avslørte transkriptomanalyser av jordbærfrukt nedregulering av viktige gener i den fenylpropanoide biosynteseveien, mens gener som koder for flavonoidtransportører og ulike peroksidaser ble oppregulert. Disse resultatene tyder på at flavonoidtransport og økt respons på oksidativt stress er viktige aspekter ved jordbærs respons på kombinerte stressfaktorer. Den siste studien avdekket en økning i konsentrasjonen av flavonolglykosider som respons på UV-C-behandling etter innhøsting. UV-C-behandling både før og etter innhøsting førte til en betydelig økning i kaempferol 3-kumaroylglukosid og quercetin 3-glukuronid, som er viktige forbindelser for plantenes fotobeskyttelse, respons på oksidativt stress og forsvarskapasitet. Til sammen viste disse responsene at UV-C-behandling både kan kontrollere fytopatogener og øke plantenes motstandsdyktighet gjennom akkumulering av fytonæringsstoffer. Diskusjonen har som hovedfokus den tidsmessige dynamikken i disse responsene, og belyser hvordan UV-C-behandling førte til kortvarige svingninger i både patogenet og verten. Ved å analysere denne dynamikken gir avhandlingen innsikt i hvordan tidspunktet for UV-C-behandlingen og organismens utviklingsstadium påvirket genuttrykk, metabolittproduksjon og fenotypiske utfall. For eksempel ble spiring forsinket ved UV-C behandling og uttrykk av melaninbiosyntetiske gener var modulert, mens behandlinger ved infeksjon på jordbærfrukt avslørte nyanserte endringer i sekundære metabolittveier, slik de som er relatert til flavonoidbiosyntese. I dette arbeidet diskuteres også potensialet for at UV-C-behandling før innhøsting kan fungere som et «priming-middel», som forbereder planten på forbedrede forsvarsreaksjoner. Selv om UV-C i vekstsesongen ikke var optimalisert for priming, tyder observasjoner av økt flavonoidakkumulering og transkriptomiske endringer på at subletal UV-C-eksponering kan øke motstandskraften mot patogener. Slike effekter kan betraktes som utilsiktede, men likevel fordelaktige biprodukter av UV-C-bruk, noe som berettiger videre undersøkelser av priming-mekanismer. Diskusjonen oppsummerer disse funnene for å foreslå hvordan UV-C-behandling kan integreres strategisk i landbrukspraksisen, slik at man balanserer umiddelbar patogenkontroll med potensielle langsiktige fordeler for planteforsvaret

Environmental risk assessment of the genetically modified vaccine Recombitek®C4

Det er ikke høyere miljørisiko ved bruk av den genmodifiserte vaksinen Recombitek®C4 enn ved bruk av tilsvarende godkjente vaksiner. Det er hovedbudskapet i en risikovurdering VKM har gjort for Miljødirektoratet. Bakgrunn Miljødirektoratet har mottatt en søknad etter genteknologiloven om bruk av vaksinen Recombitek®C4 i det nasjonale avlsprogrammet for fjellrev (Vulpes lagopus). Fjellreven er klassifisert som en sterkt truet art i Norge. Avlsprogrammet er etablert som del av bevaringsarbeidet og driftes av Norsk institutt for naturforskning (NINA). Ved avlsstasjonen i Oppdal har fire revevalper dødd av smittsom leverbetennelse (hepatitis contagiosa canis (HCC)), og man ønsker å bruke denne vaksinen for å hindre ytterligere smitte. Det finnes godkjente alternative vaksiner til Recombitek®C4, men disse ønsker man ikke å bruke fordi vaksinene inneholder et svekket (for hund), men levende valpesykevirus (CDV), som kan føre til sykdom hos fjellrev. Recombitek®C4 er pr i dag ikke godkjent i Norge eller i EU, men er blitt regelmessig brukt til vaksinasjon av hunder i over 20 år, blant annet i USA og Canada. Miljødirektoratet har bedt VKM utføre en miljørisikovurdering av vaksinen Recombitek®C4 i henhold til genteknologiloven VKMs vurdering Vaksinen Recombitek®C4 inneholder en kanarikoppevirusvektor kalt ALVAC. ALVAC er basert på Kanarikoppeviruset (CNPV), som først og fremst forårsaker sykdom hos spurvefugler, som kanarifugl. Forløpet kjennetegnes ved sykdom i hud og difteriliknende symptomer, med mulig høy dødelighet. ALVAC er en genetisk konstruert, svekket variant av CNPV, designet for bruk i vaksiner. Hverken vektoren eller viruset kan produsere eller spre nye virus mellom pattedyr. Dermed vil den mest sannsynlige veien til miljøet være via overføring til ville fugler. Avlsstasjonen i Oppdal rapporterer om en viss interaksjon mellom fjellrev og fugler, som ørn, ravn og kråke, bl.a. rundt fôrplasseringer. For å minimere mulig kontakt mellom rev og fugl, bruker man fôringsbokser og spesielle «ørnestrukturer» i innhegningene. Dersom ørn eller andre fugler likevel skulle komme i kontakt med ALVAC i perioden revene vaksineres, anses faren for smitte, sykdom og spredning blant fugler som svært usannsynlig. Flere vaksiner som inneholder ALVAC er godkjente til bruk på dyr i Norge, for eksempel i vaksiner til katt og hest. VKM konkluderer at bruk av Recombitek®C4 i det nasjonale avlsprogrammet for den rødlistede arten fjellrev ikke utgjør en økt miljørisiko sammenlignet med tilsvarende vaksiner med ALVAC-vektoren allerede i bruk for andre dyrearter i Norge. Risikovurderingen er godkjent av VKMs faggruppe for genmodifiserte organismer - legemidler.Environmental risk assessment of the genetically modified vaccine Recombitek®C4publishedVersio

A fish intestinal in vitro model for investigation of lipid metabolism and steatosis

Choline is now recognized as an essential nutrient to ensure lipid transport in Atlantic salmon. Its deficiency leads to excessive lipid accumulation in the enterocytes, a condition known as steatosis. The knowledge of lipid metabolism and steatosis in fish remains limited, motivating the use of in vitro intestinal models to perform deeper explorations. This study aimed to create an in vitro steatosis model using RTdi-MI, a new cell line derived from the distal intestine of rainbow trout. Cells were exposed to varying oleic acid (OA) concentrations over different time points (24 h, 72 h, and 168 h). Results indicated that the increasing OA concentration enhanced intracellular lipid droplet formation. Quantitative lipid analysis confirmed OA accumulation, which intensified with prolonged exposure and increased OA dose. Moreover, all cells, including controls, exhibited fatty acid metabolic activity. Such outcome was confirmed by light and fluorescence microscopy. Additionally, RTdi-MI cells expressed genes involved in lipid metabolism and synthesis similar to in vivo conditions. Collectively, our findings demonstrate the ability of RTdi-MI cells to accumulate OA in intracellular lipid droplets and mirror in vivo steatosis conditions, offering a new tool for exploring fish intestinal lipid metabolism.publishedVersio

Effects of personality, motives, and socioeconomics on hard cider consumption

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The effect of weather conditions from heading to harvest on gluten quality of spring wheat – A study of historical data 2005–2022

The gluten-viscoelastic properties are essential for breadmaking quality and are affected by both genotypes and environments, such as weather conditions. However, it is still not clear how weather conditions cause variation in gluten quality and at which stage of the grain filling they are critical. The aim of the study was to explore the relationship between weather parameters during grain filling and the viscoelastic properties of gluten. The gluten of spring wheat varieties grown over 17 seasons, resulting in a total of 70 different environments, was analyzed with the Kieffer extensibility rig. The variation in viscoelastic properties of gluten was mainly explained by environment, followed by genotype, while the genotype*environment interaction was small. The results also indicated that the periods around heading and physical maturity were the most critical when weather conditions affected the gluten quality. Our results also revealed that factors other than weather conditions are responsible for the variation in gluten quality.publishedVersio

Nye dietter og fiskehelse : evaluering av immun- og ernæringsmessige virkninger av den filamentøse soppen Paecilomyces variotii i funksjonelle fôr for Atlantisk laks (Salmo salar)

Filamentous fungi Paecilomyces variotii is an interesting microbial ingredient (MI) with high protein content and bioactive compounds such as β-glucans, mannans and nucleic acids. In the past, P. variotii has supported good growth of chicken, pigs and calves by replacing conventional ingredients. However, there is a paucity of research knowledge on the nutritional value and health benefits of P. variotii for fish. Thus, the objective of this thesis was to investigate the nutritional value and health impact of P. variotii in Atlantic salmon with focal interest on the growth performance, gut microbiota, local and systemic immune responses, as well as overall fish health and welfare in both freshwater and seawater under controlled conditions. The P. variotii used in this thesis was produced by Enifer (Espoo, Finland) on sulphite stillage through continuous fermentation and dried at 70 0C using fluidized bed. Regarding the articles related to this thesis, in paper I, we used a desk study method to review state-of-the-art literature in which in vitro salmonid models have been used to study microbial derived immunostimulants (β-glucans, LPS, dsRNA/poly[I:C]) and economically relevant salmonid pathogens (viruses, bacteria, parasites). We identified their strength and weaknesses and provided future perspectives on 3D models like bioreactors, organ-on-chip, organoids etc. Only partial information is obtained from current in vitro models (mostly 2D); hence they cannot be proxies or direct replacements but suitable complements of in vivo models. The study found several 3D models, where research into which are advanced in mammals but not fish. Future 3D models like gene editing, bioreactors, organ-on-chip models and organoids are interesting models that can be adapted for studies related to fish. Like the 2D models, 3D models in their current state cannot individually replace live animal testing. However, a combination of the 3D models and advanced bioengineering can enhance their usage as possible animal replacement models. In paper II we characterized the bioactive composition of P. variotii, its bioactivity on primary cultures of salmon head kidney leucocytes (HKLs) and spleen leucocytes (SLs) with or without heat inactivated M. viscosa (an important salmonid pathogen), as well as the impact of downstream processing (DSP) on the ultrastructure of P. variotii. We also investigated the impact of carbon source (sulphite stillage, glucose or glycerol) and choice of fermentation (batch or continuous) on the crude protein (CP) and glucan composition of P. variotii. Glucose or glycerol in combination with batch fermentation significantly increased the glucan but lowered CP contents of P. variotii and vice versa for sulphite stillage together with continuous fermentation. Drying also significantly reduced the thickness of the cell wall and mannan layer of P. variotii which could in turn improve digestibility and access to nutrients. Stimulating HKLs with different fractions of P. variotii induced an early pro-inflammatory (tnfα, il1β) and anti-microbial response (cath2) while in SLs, both pro- and anti-inflammatory response (tnfα, il6 and il10) was induced. Stimulating HKLs and SLs with M. viscosa alone upregulated pro-inflammatory cytokines (tnfα, il1β, il6), antimicrobial response (cath2), and receptors (sclra, sclrb) but suppressed regulatory response (il10). Prolonged co-stimulation (48h) with M. viscosa and P. variotii induced homeostasis (tgfb, il6) and antimicrobial (cath2) response in SLs. In paper III, to understand the impact of P. variotii on short-term growth performance and health responses, vaccinated Atlantic salmon pre-smolts were fed diets where P. variotii replaced CP from fishmeal (FM), wheat gluten meal (WGM), and soy protein concentrate (SPC) at 0%, 5%, 10% or 20% for 28 days in freshwater (FW). The mycoprotein supported the short-term growth performance of Atlantic salmon and induced a dose-dependent linear improvement of FCR with increasing inclusion of P. variotii. High CP replacement with P. variotii (20%) induced downregulation of receptors and cytokines (sclra, sclrc, cr3, il1b, tgfb) indicative of truncation of signal transduction in the distal intestine (DI). In the head kidney (HK), P. variotii activated and controlled immune response through c-type lectin receptors, signal molecules, cytokines, effector molecules, and coordinated communication between innate and adaptive immune response through antigen presenting cell markers (mhcii, cd83) and T cell transcription factors (gata3, rorc, foxp3). Interestingly, the HK proteomic profile of fish fed the 20% P. variotii diet showed reduced proteins related to inflammation (apoptosis, necroptosis etc), and signalling (TLRs, CLRs etc) contrary to the gene expression data. However, these proteins were increased in the skin mucus with other key proteins related to the complement activation (C1q, C4, C7), arginine and proline metabolism. Fish fed diets with CP replacement with 5% P. variotii showed high titres of specific IgM against V. anguillarum in the serum. Finally, 5% and 10% CP replacement with P. variotii increased both the α- and β-diversity and modulated lactic acid bacteria (LAB), including Ligilactobacillus. In paper IV, the fish from freshwater were transferred to saltwater (SW) and fed for additional 35 days with the same diets as in FW (paper III). The results indicate that P. variotii supports the short-term growth performance of Atlantic salmon post-smolts. Increasing inclusion of P. variotii in diets increased the DI simple fold height but reduced the area of epithelium occupied by goblet cells (GCs). Again, 5% CP replacement with P. variotii decreased the average size of supranuclear vacuoles in the DI of the fish. In addition, 20% CP replacement with P. variotii increased serum and skin mucus total IgM titres while 5% CP replacement with P. variotii increased epidermal muc-like proteins and cytokine il10 in the HK of the fish compared with the control diet group. We also observed a significantly lower serum creatine kinase (CK) in fish fed the 20% CP replacement diet compared with the control diet. Interestingly, we observed a high occurrence of welfare disorders (e.g., scale loss, fin rot/damage, skin haemorrhage) in fish fed the 20% CP replacement with P. variotii. Based on the results, it can be concluded that P. variotii is a promising novel MI whose nutritional and functional composition can support fish growth and meet the prophylactic needs of salmonids. Thus, beyond the nutritional value of P. variotii, its functional potential can be harnessed through immuno-nutritional strategies to improve the health and welfare of farmed salmonids in sustainable and eco-friendly manner

Explaining the recent reduction of Indonesia’s deforestation

Executive summary: Deforestation in Indonesia has declined sharply since 2015-2016. We use a combination of spatial land use change data, interviews with stakeholders and statistical analysis to explain this reduction. The exact extent of the reduction in deforestation depends on the data set used, and the years or time periods compared, varying between 40% and 90%. A robust estimate is that deforestation has fallen by at least 50% since ca. 2016. The decline is uneven across islands and provinces. The two main “deforestation islands” Kalimantan and Sumatra experienced a major decline, with a relatively larger decline in the former. The provinces with the largest absolute decline were Riau, South Sumatra and Central Kalimantan, followed by West and East Kalimantan. In terms of commodities driving deforestation (“direct drivers”), palm oil is still the most important commodity (46% for 2018-2022, according to MapBiomas Indonesia data, compared to 55% for the period 2010-2017). Pulp is on the rise as a deforestation driver, particularly in Kalimantan. In the three high deforestation provinces on that island, the demand for pulp was behind almost 1/3 of the natural forest conversion in 2022. Mining for coal and valuable minerals such as nickel is also on the rise, with MBI data suggesting its share of national deforestation being 5.4% (2021-2022), compared to less than one (0.9%) for the 2010-2017 period. Mining is particularly important in Sulawesi, where it according to one estimate accounted for 30% of the deforestation in 2021-2022. Five hypotheses are put forward to explain the reduction, related to public policies, private (corporate) policies, civil society pressure, commodity prices, and forest scarcity. New public (government) policies have been a main reason for the decline. The moratorium of new permits of primary forest and peatlands in 2011 has had an impact, but also took time to produce an effect on the ground effect (in part as it was a ban on issuing new licences). Interviewees also stressed that better sectoral coordination related to the moratorium and other reforms has been key for the slowdown. A second set of reforms relate to fire management and peatland protection, sparked by the devastating 2015 forest fires. The interviewees highlight this as a key policy reform by, for example, increasing the accountability of subnational government officials for fire management. Results-based payments (RBP) or result-based contributions were central in the Letter of Intent between Indonesia and Norway in 2011 and in the new MoU of 2022, although the first payment was not made before in 2022. An RBP-based project of more than USD 100 million was approved by the Green Climate Fund in 2020. RBP has also been implemented at subnational level starting in East Kalimantan and Jambi with external donor support. Some reports indicate positive results, although they are likely to be too location-specific and came too late to have had major impacts on the observed decline in national deforestation figures since 2016. Private regulation such as certification and corporate pledges show promising signs with an increasing share of oil palm plantations being certified. Yet, breached are reported, and the forest encroachment factor (i.e., the share of new land being converted from forest) has not dropped as much as to be expected. Moreover, certification of pulp production and mining is lagging behind. Civil society pressure has played a role in particularly two areas, although the exact role and contribution are hard to assess. First, CSOs are active actors on the policy arena, also to influence private sector initiatives and policies. Second, CSOs are important watchdogs for both implementation of public and private regulations and pledges. This has helped “bringing the forests to the court”, as one interviewee observed. Key commodity prices were relatively stable during the 2016-2020 period, and thus cannot explain much of the decline. Yet, no major increase in the prices of deforestation-risk commodities made the implementation of both public and private policies less costly (both for politicians and producers), and made violations of laws and regulations less profitable, increasing policy effectiveness. The prices of coal and minerals such as gold and nickel have, however, increased steadily over the period. The nickel price quadrupled between 2016 and 2022. The increasing role of mining in deforestation can largely be explained by the high and increasing profitability, and demand is likely to grow steadily in the coming years due to the global energy transition. The statistical analysis suggests that up to 1/3 of the reduction in deforestation can be explained by forest scarcity (forest transition). When a province hits ca. 40% forest cover, deforestation tend to decline. While we conclude that all five hypotheses put forward are relevant to explain the recent decline in deforestation, we tentative conclude that public polices – in combination with a forest transition (scarcity) effect – has been the most important factors. Private policies show more mixed results. Behind the public and private policy changes, civil society has played a key role in policy reforms and in promoting more effective implementation though its watchdog role. Non-increasing agricultural commodity prices have made the implementation of forest conservation policies less costly. Deforestation is still a profitable activity for land users and (sub)national governments, and forest conservation is a continuous battle, with future challenges emerging: the effectiveness of current policies may weakened over time, political priorities may change, prices of deforestation-risk commodities may rise, and the composition of direct drivers change – requiring a shift in the policy focus.   Recommendations 1. Be ahead of the curve: The forest transition suggests a natural development with decreasing deforestation in low forest cover regions (provinces) and increasing deforestation in high forest cover regions. The major future deforestation threats have moved eastwards in Indonesia. Mining is likely to be a key driver of deforestation in Sulawesi, while food estates, oil palm and pulpwood plantations in Papua are emerging as direct drivers in new deforestation hotspot. 2. Incentives for high-forest, low-deforestation areas: Much focus in the REDD+ discussion has been on reducing emissions from high-deforestation areas, and rightly so. Taking a more long-term view and preventing future increases in deforestation, mechanisms that incentivise the conservation of high-forest, low-deforestation areas are also needed, while noting the challenges of estimating additionality – particularly if carbon credits from these areas enter carbon trading. 3. New deforestation-risk commodities: The deforestation debate in Indonesia has traditionally focussed on timber logs and palm oil. Timber production is in decline, and the share of palm oil-driven deforestation is also declining, although oil palm cultivation is still the main immediate land use after forest clearing. Demand for biodiesel based on palm oil may make it maintain its role as the no. 1 deforestation-risk commodity. Pulp and mining (nickel) have increased its share of direct deforestation drivers, and with pricing of key minerals on the rise this pressure is likely to continue. Plans for large food estates is also likely to make it an increasingly important driver. 4. The energy transition and deforestation: The global energy transition has increased the demand for both renewable energy (palm oil and wood pellets) and minerals (such as nickel). Paradoxically, these also pose a threat to Indonesia’s natural forests, and balancing these trade-offs remains a major challenge both for policy makers and advocacy groups. 5. CSOs watchdog role important for effective implementation: The private sector initiatives are commendable, but their impact hinges on their effective implementation. Clearing new forest land remains a profitable option from a business perspective, and without clear sanctions of violations (both formal and informal such as reputational risk) illegal and semi-illegal forest conversion is likely to continue. 6. Better data on “other agriculture” commodities: The share of “other agriculture” as a direct driver is increasing, yet this remains a black box in many data sets and analyses. More data on what constitutes this driver and their relative importance (including cocoa, coconut, coffee, rice, rubber sugar) are needed to design and implement targeted policies, also related to the EUDR. 7. Transparency in data sets and their uses: Several data sets on deforestation or tree cover loss are available, with quite different primary data sources and forest/deforestation definitions. This creates a wide range of figures, opening for cherry picking and selective uses. Full transparency on definition and data transformations is needed to enable better comparison and detect underlying trends

Utvikling av anaerob høytetthetsdyrking for bærekraftig produksjon av mikrobielt protein ved hjelp av denitrifikasjon

Future food production faces the immense challenge of feeding a growing population with an increasing demand for resource-intensive food in a sustainable way. The high environmental footprint of conventional food production opens the floor to alternative sources of food and feed, with single-cell protein (SCP) emerging as a promising solution. SCP offers a high protein content (up to 80%), favorable amino acid profiles, and the presence of carbohydrates, vitamins, fats, and antioxidants increasing the nutritional value. The production is independent of seasonal and climate variations, has low requirements for arable land and resources, and has low greenhouse gas emissions. SCP is generally produced by high cell density cultivations (HCDCs) to maximize the volumetric yield. However, conventional HCDC relies on aerobic respiration, which is limited by the low solubility and diffusion rate of oxygen in liquid. Oxygen limitations may result in anoxic zones accompanied by fermentation and accumulation of unwanted byproducts inhibiting growth. Denitrification, the stepwise reduction of nitrate (NO3-) to nitrogen (N2) via nitrite (NO2-), nitric oxide (NO), and nitrous oxide (N2O), is the most efficient form of anaerobic respiration and overcomes the rate-limitations inherent in aerobic respiration due to the high solubility of NO3- and NO2-. Previous attempts at HCDC with denitrification most likely failed due to denitrification-driven increases in pH necessitating tight pH control and the provision of NO3- as a salt resulting in salt accumulation. This work presents a novel method for anaerobic HCDC that overcomes these challenges by using nitric acid (HNO3) as both the pH regulator and the provider of NO3- for denitrification. The bioreactor is operated as a pH-stat where denitrification-driven increases in pH trigger HNO3 injections, which in turn control the provision of the carbon source from a separate feed pump. The study was initiated by proof-of-concept experiments that demonstrated the viability of the method by growing the model denitrifier Paracoccus denitrificans Pd1222 to a density of more than 20 g dry weight L-1 (Paper I). We were able to reach the expected yield based on the provided substrate, but the growth rate was much lower than what we observed in low-density batch cultures. In addition to the low growth rate, we observed 1) the accumulation of polyhydroxyalkanoates (PHA), a storage and energy reserve typically encountered during carbon excess, and 2) a low expression of NirS, the enzyme responsible for the reduction of NO2- to NO. These challenges were addressed in subsequent experiments where we aimed to develop the method further (Paper II). We switched to Paracoccus pantotrophus GB17, a closely related strain with a higher NirS pool, that was also shown to adapt better to the bioreactor environment. The compositions of the feed solutions were refined to prevent accumulation of byproducts, including PHA, and improve the growth conditions. We incorporated ammonium nitrate (NH4NO3) as the nitrogen source to remove the cost of NO3- assimilation and designed new acid solutions with minerals and trace elements. We hypothesized that the slow growth rate was due to the high concentration of carbon dioxide (CO2) in the liquid lowering the pH, thereby interfering with the provision of the substrates which is triggered by alkalization. This was investigated through in silico simulations of the NO3-, CO2, and pH kinetics in the bioreactor and rapid sampling of the bioreactor. The modeling and empirical data showed that the NO3-, which was injected at intervals of several minutes, was depleted within seconds. We aimed at improving the CO2 removal by dynamically changing the sparging flow rate and pH setpoint in response to changing partial pressure of CO2 in the off-gas and experimenting with lower pH limits. Although the process is still limited by a slow growth rate, we were able to reach densities of more than 60 g dry weight L-1 using P. pantotrophus. The biomass was analyzed and shown to have an average protein content of 75%. While anaerobic high cell density cultivation is proven feasible, one major challenge for the upscaling is the high cost of HNO3. We have therefore validated HNO3 produced by N2 Applied using plasma technology, which is expected to become a cheaper and more sustainable alternative in the future (Paper III). Biomass produced using our process was also tested by the Center for Feed Technology (Fôrtek, Norwegian University of Life Sciences, Norway) in successful feed trials with Atlantic salmon. This demonstrates that SCP produced by our process can be used as a protein source in aquaculture feed. The work presented in this thesis bridges fundamental research on microbial physiology with applied science, demonstrating the proof-of-concept, development, and commercial potential of a novel method for anaerobic high cell density cultivation. Although only tested for heterotrophic growth on glucose, the process has the potential to be adapted to a wide range of strains, substrates, and metabolic strategies. This work paves the way for anaerobic high cell density cultivation as a supplement to existing methods, advancing the field of sustainable food production.Fremtidens matproduksjon står overfor den enorme utfordringen å skulle fø en voksende befolkning med en økende etterspørsel etter ressurskrevende mat på en bærekraftig måte. Det høye miljøavtrykket til matproduksjon åpner for alternative kilder til mat og fôr, hvor mikrobielt protein (SCP) fremstår som et godt alternativ. SCP har et høyt proteininnhold (opptil 80%), gunstig aminosyreprofil, og karbohydrater, vitaminer, fett og antioksidanter som øker den ernæringsmessige verdien. Produksjonen er uavhengig av sesong- og klimavariasjoner og har lave krav til dyrkbar mark og ressurser, samt lavt klimagassutslipp

Vomsentrert sammenstilling av storfeholobionten

The holobiont perspective takes into account the complete system of an animal including its microbiomes and to some extent the factors that form its external environment. This view has a scientific motivation: In the case that the microbiome affects the host, studying the host in isolation severely limits access to the complete information needed to understand the biology of the host. As a model for such a “holobiont” system, a starting point was taken in cattle and focused on the rumen and the interface that connects its inherent microbiome to the metabolism of the host. As the rumen microbiome produces a vital component of metabolites that the host budgets for energy and nutrient assimilation, it has a wide potential to impact the host. The association between the host and its rumen microbiome has made it a focal point for modulation strategies to improve health, nutrition and sustainable production of ruminants. However, the complexity of the rumen microbiome and its interactions with the host represent major challenges that must be overcome before microbiome-based approaches can be used in practice. To improve reconstruction of the rumen microbiome, a high-resolution dataset was generated for deep analysis from 80 cattle subjected to a feedlot trial. Here, the rumen microbiome was sampled over time, and host tissue (rumen wall and liver) samples were collected upon sacrifice, after rigorous measurement of the cattle’s key performance traits (KPTs) and methane emissions. To study the ruminant holobiont, molecular layers in both the host and its rumen microbiome were reconstructed. Multiple molecular layers (DNA, RNA, protein, metabolites, and glycans) as well as the host phenotype were analyzed, in order to track how potential interactions affected metabolism in 24 individual animals that exhibited the highest natural variation in measured methane yield. As most biological variation of an organism is encoded in the genome, DNA sequencing is central to forming a foundation upon which to assemble the holobiont. To further enhance our DNA analyses, long-read and high accuracy short-read shotgun metagenome sequencing was applied to reconstruct the microbial genomes of the rumen microbiome. To track which genes were expressed, transcriptomics was applied, and to analyze the presence of translated proteins and their derived metabolites, also proteomics and metabolomics. For complex eukaryotic populations that are unamenable to shotgun sequencing approaches, such as the protozoa and fungi, genomes were sourced from collaborative projects. Analyzing a single molecular layer requires a specific set of technical tools. For this purpose, it is described how microbial genomes can be reconstructed, and how their relevant metabolic functions can be identified. Specifically in relation to the carbohydrate-active enzymes (CAZymes) that enable ruminants to assimilate carbon and energy from plant fibers, representing the basis for the energy budget of the host. As it was not possible to identify a suitable pipeline with the tools necessary to analyze and compare the metabolic function of the archaeal and bacterial genomes generated in our datasets, an easy to use platform for analyzing metagenome-assembled genomes (MAGs) was developed. This pipeline is now distributed on Bioconda as CompareM2. To apply the wide tool set that was put together and attempt to improve resolution and general understanding of how the ruminant host and its microbiome function as an integrated unit, an experimental cattle holobiont dataset was analyzed. The sampled molecular layers were refined to become biologically relevant representations on which integrative holo-omic methods could be applied to identify and investigate possible host-microbiome interactions. In practice, simpler computational dimensionality reduction methods may offer greater interpretability and allow more direct biological interpretation than more complex computational methods. Applying these methods to our experimental data led to the discovery that the protozoal fraction of the rumen seemingly drives two exclusive community types across the individual animals that were sampled, which have previously been described from micrography and 18S studies. These are referred to as RCT-A and -B (rumen community type). RCT-B is dominated by protozoa affiliated to Epidinium spp. that were observed to employ a plethora of fiber-degrading enzymes, which most likely provide favorable conditions for saccharolytic bacteria such as Prevotella spp. Conversely RCT-A is dominated by Isotricha and Entodinium protozoal species and harbors a wider representation of fiber, protein and amino acid fermenters. While no clear host effect for these rumen community types is found, there are signs in the more complex network analysis based computational methods that certain microbial populations of Acutalibacteraceae prevalent in RCT-A affect methionine metabolism in the rumen wall. This calls for further refinement of the holo-omic analyses and biological characterization. Finally, our work highlights the need for de facto standards to refine individual molecular layers, and to find common methods for data integration across these molecular layers that represent the host-microbiome axis.Holobiont-perspektivet tar hensyn til det komplette systemet til et dyr, inkludert dets mikrobiomer og, til en viss grad, faktorene som danner dets ytre miljø. Dette perspektivet har en vitenskapelig motivasjon: I tilfeller der mikrobiomene påvirker verten, begrenser det å studere verten i isolasjon tilgangen til den komplette informasjonen som er nødvendig for å forstå vertens biologi. Som en modell for et slikt holobiont-system har vi tatt utgangspunkt i storfe, med fokus på vomma og grensesnittet som forbinder dets iboende mikrobiom med vertens metabolisme, spesifikt vomveggen og leveren. Siden mikrobiomet i vomma produserer en viktig komponent av metabolitter som verten bruker til energibudsjettering og næringsopptak, har det et stort potensial til å påvirke verten. Sammenhengen mellom verten og dens iboende vommikrobiom er derfor et fokusområde for moduleringsstrategier for å forbedre helse, ernæring og bærekraftig storfeproduksjon. Vommikrobiomets kompleksitet og dets interaksjoner med verten representerer imidlertid store utfordringer som må takles før mikrobiom-baserte tilnærminger kan brukes i praksis. For å forbedre rekonstruksjonen av vommikrobiomet har vi generert et høyoppløselig datasett for dyp analyse fra 80 storfe som ble eksponert for en fôringsprøve. Her ble vommikrobiomet tatt prøver av over tid, og prøver fra vomveggen og leveren ble samlet ved avliving, kort tid etter grundige målinger av storfeets nøkkelprestasjonsegenskaper, inkludert metanutslipp.Novo Nordisk Foundation ; NORBISpublishedVersio