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Abstract Wolfiporia cocos, a versatile fungus acclaimed for its nutritional and therapeutic benefits in Traditional Chinese Medicine, holds immense potential for pharmaceutical and industrial applications. In this study, we aimed to optimize liquid fermentation techniques and culture medium composition to maximize mycelial biomass (MB) yield, pachymic acid (PA) concentration, and overall PA production. Additionally, we investigated the molecular basis of our findings by quantifying the expression levels of genes associated with PA and MB biosynthesis using quantitative real-time polymerase chain reaction. Under the optimized fermentation conditions, significant results were achieved, with maximum MB reaching 6.68 g l−1, PA content peaking at 1.25 mg g−1, and a total PA yield of 4.76 g l−1. Notably, among the four examined genes, squalene monooxygenase, exhibited enhanced expression at 0.06 ratio under the optimized conditions. Furthermore, within the realm of carbohydrate-active enzymes, the glycoside hydrolases 16 family displayed elevated expression levels at 21 ratios, particularly during MB production. This study enhances understanding of genetic mechanism governing MB and PA production in W. cocos, highlighting the roles of squalene monooxygenase and glycoside hydrolases 16 carbohydrate-active enzymes.

Abstract We focused on the production of docosahexaenoic acid (DHA)-containing microbial lipids by Aurantiochytrium sp. using of defatted soybean (DS) as a nitrogen source. Defatted soybean is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytrium sp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytrium sp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytrium sp. 6-2 showed 15.8 ± 3.4 g/L DHA productivity (in 54.8 ± 12.1 g/L total fatty acid production) in 1 L of the FDS medium. Therefore, DHA produced by Aurantiochytrium sp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.

Formate production via both CO2 reduction and cellulose oxidation in a solar-driven process is achieved by a semiartificial biohybrid photocatalyst consisting of immobilized formate dehydrogenase on titanium dioxide (TiO2|FDH) producing up to 1.16±0.04 mmolformate gTiO2-1 in 24 hours. Isotopic labelling experiments with 13C-labelled substrates support the mechanism of stoichiometric formate formation through both redox half-reactions. TiO2|FDH was further immobilized on hollow glass microspheres to perform more practical floating photoreforming allowing vertical solar light illumination with optimal light exposure of the photocatalyst to real sunlight. Enzymatic cellulose depolymerization coupled to the floating photoreforming catalyst generates 0.36±0.04 mmolformate mirr-2 after 24 h. This work thus presents simultaneous solar-driven valorization of waste streams, demonstrates the advantages of biohybrid photocatalysts in photoreforming for the first time and will provide inspiration for the development of future semi-artificial waste-to-chemical conversion strategies.

Abstract Thermo-acidic pretreatment of lignocellulosic biomass is required to make it amenable to microbial metabolism and results in generation of furfural due to breakdown of pentose sugars. Furfural is toxic to microbial metabolism and results in reduced microbial productivity and increased production costs. This study asks if deletion of yghZ gene which encodes a NADPH-dependent aldehyde reductase enzyme results in improved furfural tolerance in Escherichia coli host. The ∆yghZ strain—SSK201–was tested for tolerance to furfural in presence of 5% xylose as a carbon source in AM1 minimal medium. At 96 h and in presence of 1.0 g/L furfural, the culture harboring strain SSK201 displayed 4.5-fold higher biomass, 2-fold lower furfural concentration and 15.75-fold higher specific growth rate (µ) as compared to the parent strain SSK42. The furfural tolerance advantage of SSK201 was retained when the carbon source was switched to glucose in AM1 medium and was lost in rich LB medium. The findings have potential to be scaled up to a hydrolysate culture medium, which contains furan inhibitors and lack nutritionally rich components, under bioreactor cultivation and observe growth advantage of the ∆yghZ host. It harbors potential to generate robust industrial strains which can convert lignocellulosic carbon into metabolites of interest in a cost-efficient manner.

The transition to cleaner energy sources is fundamental for mitigating the effects of climate change. One promising example is lignocellulosic biomass, a renewable and sustainable energy source that serves as a precursor to various compounds, including γ-valerolactone (GVL). GVL can be utilized as a biofuel or biopolymer. This project evaluates the potential use of SrFe12O19/Ni-P and graphite/Ni-P as catalysts for the photo-thermocatalytic and cost-effective production of GVL from levulinic acid, using a laser light as a source of energy. The goal is to scale up this process for future industrial applications. The catalysts were functionalized via a Ni-P electroless deposition process. The impact of temperature, reaction time, and electroless deposition coating time on catalyst efficiency was analysed. For the effective catalysts, different coverages thicknesses were tested. Optimal experimental conditions for reactor experiments with a laser dispositive were previously established by conducting autoclave tests in a conventional oven. Autoclave tests results evidenced that the SrFe12O19/Ni-P material did not exhibit catalytic properties for the production of γ-valerolactone. In contrast, graphite/Ni-P catalysts achieved nearly 100% conversion, with an optimum minimum reaction temperature of 120 ºC. To evaluate the potential of the graphite/Ni-P catalyst as a photo-thermal catalysts for γ-valerolactone production, experiments were conducted using the laser apparatus under the aforementioned conditions. The results showed that the graphite/Ni-P catalyst with a 10-minute deposition time exhibited higher conversion rates at lower reaction times and demonstrated greater durability and stability throughout the reusability cycles. Consequently, the graphite/Ni-P catalyst with a 10-minute deposition time was identifies as a suitable catalyst for GVL production. Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2024, Tutors: Albert Serrà Ramos, Elvira Gómez Valentín

To decrease our society’s dependence on polluting fossil resources, alternative sources for chemical and fuel production need to be developed. Organic residual streams are a renewable feedstock that can be used to replace these fossil-based fuels and chemicals. In this thesis bioelectrochemical chain elongation (BCE) has been studied to convert short chain fatty acids (SCFAs, model substrates for acidified organic residual streams) into biobased intermediate chemicals. BCE is subtype of a microbial electrosynthesis (MES) system, in which microorganisms catalyse the elongation of SCFAs and/or CO2 towards medium chain fatty acids in an electrochemical cell.Part 1 of this thesis studied the formation of valuable products from SCFAs using BCE systems. In chapter 2 it started with the proof of concept of using an electrode for the sustained chain elongation of CO2 and acetate in continuous BCE systems. Four BCE reactors were used to study the role of applied current: two were applied with 3.1 A m-2 (projected surface area of electrode) and the other two with 9.4 A m-2. n-Butyrate (nC4) was the main identified product in all reactors. The highest applied current led to the highest nC4 production rate of 0.54 g L-1 d-1 (24.5 mMC d-1). The highest concentration of nC4 reached under high current regime was 0.59 g L-1 (26.8 mMC). Trace amounts of propionate and n-caproate were also produced, but no alcohols were detected over the course of the experiments (163 days).To improve BCE and enhance production, in chapter 2 as well a literature review is provided to give insights into all the reported pathways to produce nC4 in fermentations. In fermentative chain elongation soluble electron donors, like ethanol or lactate, supply reducing equivalents and drive microbial metabolism. Since such compounds were not detected in the BCE reactors, it was hypothesised that nC4 production was limited by intermediate production and subsequent fast consumption of ethanol or lactate.This hypothesis of intermediary production of ethanol or lactate limiting BCE performance was verified in chapter 3. Both ethanol and lactate were separately introduced in triplicate BCE reactors applied with 9.4 A m-2. Both compounds did not significantly affect the rates of nC4 production. Next to these compounds, the effect of formate on nC4 production was tested. Formate injection led to acetate production and decreased nC4 production. The results suggested that formate conversion to acetate competed with acetate elongation to nC4 for electrons. This competition subsequently resulted in decreased production of nC4. To investigate role of the electrode as electron donor, the current was increased to 18.1 A m-2. This increase in applied current doubled the production rates of nC4. Hence, this chapter demonstrates that the nC4 production in our BCE systems was not limited by intermediate production of well-known electron donors, but was driven by electrode-derived electrons.For BCE to become a feasible organic waste valorisation technology, the studied substrate range needs to extend beyond acetate reduction. Therefore, in chapter 4 four different substrate feeding strategies and the subsequent product spectrum were investigated: I) acetate, II) acetate and propionate, III) acetate and n-butyrate, and IV) a mixture of acetate, propionate and n-butyrate. In phase I, nC4 was produced at 0.9 g L-1 d-1 (39.7 mMC d-1). After introduction of propionate in phase II, n-valerate (nC5) production started and sustained until medium was changed at the start of phase III. The maximum concentration of nC5 reached was 1.2 g L-1 (60.6 mMC), and the highest production rate was 1.1 g L-1 d-1 (57.5 mMC d-1) at a high carbon-based selectivity of 73.8 %. This seems contradictory to ethanol chain elongation studies in which acetate is concurrently formed leading to straight fatty acids as by-products. Upon introduction of acetate and n-butyrate, n-caproate (nC6) production started and reached a maximum concentration of 0.3 g L-1 (15.8 mMC). The nC6 formation selectivity was 83.4 % in phase III. When all the three SCFA were supplied as substrate in phase IV, nC5 was the main product (95.4%). The observed preference for propionate elongation over both nC4 formation or nC6 formation is in contrast to fermentative ethanol-based chain elongation studies.Part 2 of this thesis focusses on the extraction of the bioproducts from dilute aqueous streams using ionic liquids. The conversion of organic waste streams as renewable feedstocks into carboxylic acids (such as SCFAs but as well the medium chain fatty acids (MCFAs)) results in relatively dilute aqueous streams. These relatively low concentrations are a major bottleneck for these bioprocesses to compete with the production of platform chemicals based on fossil resources. A way to overcome this bottleneck is to extract the carboxylates from the fermentation broths using liquid- liquid extraction. Hydrophobic ionic liquids (ILs) are novel extractants which can be used for this purpose. Ionic liquids are salts comprised of ions, with relatively low melting temperatures (often below 100°C). By varying the types of ion and, for example, the branching of the ions, the physical properties of the IL, such as its hydrophobicity, can be tailored. To integrate these ILs as in situ extractants in biotechnologies, the ionic liquid should be compatible with the bioprocess.In chapter 5 the biocompatibility of the two hydrophobic ILs [N8888][oleate] and [P666,14][oleate] were investigated in a two-phase system (IL layer on top of water phase). Commonly, ILs are synthesized in organic solvents, such as toluene and ethanol. After synthesis some trace amounts of these solvents can remain in the IL. When that hydrophobic IL is placed on top of a water phase, the trace amounts of synthesis solvent can leak into the water phase. To circumvent possible toxic effects of the trace amounts of solvent in the IL, water was used as synthesis solvent. After synthesis of the two ILs, their bioprocess compatibility was assessed. Methanogenic granular sludge was placed in medium without carbon source, and on top of that medium the IL phase was placed. After 24 days the sludge was separated from the water phase and placed into fresh medium. Upon transfer of the sludge into fresh medium with acetate as substrate, [P666,14][oleate] exposed granules were completely inhibited. Granules exposed to [N8888][oleate] sustained anaerobic digestion activity, although moderately reduced. Co-ions of the starting materials of the ILs, bromide and oleate, could have remained in the IL after synthesis. Both bromide (5 to 500 ppm) and oleate (10 to 4000 ppm) were demonstrated to not inhibit methanogenic conversion of acetate. Conclusively, [P666,14] was identified as a bioprocess incompatible component and [N8888][oleate] as bioprocess compatible.For an IL to become the envisioned in situ extractants for bioprocesses, the IL needs to be regenerated and reused. In chapter 6 a concept of an IL as transport liquid is presented, in which a product (from a bioprocess) is in situ extracted into a hydrophobic IL. The subsequent extraction of the product from the IL (i.e. regeneration) does not necessarily need to take place in/at the same physical location, time and/or medium as where the extraction of product into the IL occurred. Therefore, the IL can be regarded as transport liquid of the product.To study the feasibility of this concept, the bioprocess compatible hydrophobic IL [N8888][oleate] was used for two successive cycles of i) extraction of SCFAs into the IL [N8888][oleate] and ii) regeneration of the IL. For the regeneration of the IL a novel method was described which employs microorganisms to assist in IL regeneration, naming it ‘microbial assisted regeneration’. Microbial assisted regeneration is beneficial as no additional salt is needed for both pH control of the bioprocess as well as for recovery of the products from the IL. The experiments in this chapter demonstrate the potential of using hydrophobic ILs as transport liquid between two bioprocesses. When the concept of an IL as transport liquid is coupled with the proposed microbial regeneration method, two distinct biological processes can be coupled.For BCE to become an industrial waste valorisation technology, the production needs to be improved. Although the electron transfer pathways are not unravelled yet, chapter 7 gives an overview of all the nowadays described pathways. In this way, the coupling of microbial metabolism with an electrode can be understood more. Based on these insights, several recommendations are provided to improve BCE and to render the technology mature enough to prove its potential using real acidified organic residual streams.

Full data table for Carbon content, Nitrogen content, and biovolume measurements of planktic Rhizaria. This data is associated with the publication: Mansour, J.S., Norlin, A., Llopis Monferrer, N., L'Helguen, S., and Not, F. (2020). Carbon and nitrogen content to biovolume relationships for marine protists of the Rhizaria lineage (Radiolaria and Phaeodaria). Limnol. Oceanogr. doi: 10.1002/lno.11714 Rhizaria are ubiquitous in oceanic waters. They have previously been underestimated due to their fragility, but recent (molecular) studies show that they are major components of the planktic community and contribute greatly to, among others, the carbon and silica flux. This study provides key data on the carbon and nitrogen content of Rhizaria and its relation to cell biovolume. Here presented are the raw data of measured (CN Elemental Analyzer) carbon and nitrogen content of Rhizaria (i.e. specifically Collodaria, Acantharia, Nassellaria, Spumellaria, Aulacantha, Protocystis, and Challengeria), as well as picture-based estimates of biovolume and derived characteristics. The biovolume-to-mass relationship for these protists has allowed the formulation of mass-to-volume equations and ratios that can be used to estimate mass from biovolume. Our data shows that global Rhizaria biomass might have in fact still been underestimated. This will be pivotal information for studies of ocean ecology and for modeling biomass fluxes, whether it is biogeographic or smaller-scale system dynamics.

The main aim of study is examing farmers understanding towrds the emerging concepts of sustainable agricultural waste management falls under circular agriculture-a circular, green economy with cleaner production and bioeconomy reconciled with economic, social and environmental dimensions. This study is a prototype in Mirab Gojjam Zone of Amhara region as well in Ethiopia. we seek to determine farmers’ willingness-to-pay (WTP) for eco-friendly AWM. Data were collected through a contingent valuation survey of 353 randomly selected farmers in Mirab Gojjam. The improper discarding and unsafe use of agricultural waste causes health and environmental problems. It also contributes to GHG emissions, particularly in Mirab Gojjam, the focus of our study. We thus assessed farmers’ WTP for eco-friendly AWM to improve the local environment in Mirab Gojjam and maintain low carbon sustainable development. The result indicates an annual WTP of 6.84 household days (equivalent to 273.5 Birr) for the surveyed farmers. The Tobit results indicate that the social capital, socioeconomic, cognitive, and altruism variables significantly influence the degree of WTP. Our results are thus useful for understanding farmers’ attitudes and WTP for eco-friendly AWM, as well as the need for private and public instruments in agricultural waste for developing and disseminating resource technology.To improve the use of agricultural waste in biofuel, construction, and feedlot and to enhance sources of rural income, green marketing and low-carbon innovative recycling technologies should be established parallel to proper discarding. In our study, the mean WTP in labour days per year was higher than the amount of money farmers that were willing to pay for eco-friendly AWM.We thus propose three suggestions for sustainable and eco-friendly AWM. First, the government must assess and understand the current generation potential of agricultural waste. It must formulate rural sustainable waste management policies and create awareness accordingly.Second, agricultural waste recycling technology should be provisioned in rural areas through public–private collaboration. The vacuum in the local waste recycling market should be addressed by opening the market to private and informal micro-enterprise sectors, along with full government support.Lastly, proper follow-up and inspection is important to ensure all parties function in accordance with the laws, norms, and policies.Generally speaking, AWM must be prioritised as much as other SDGs to sustain holistic development, cleaner production, and clean-living environments for reducing carbon emission and looping conventional waste into circular agriculture.