Polyhydroxyalkanoates (PHA) are a family of biodegradable polyesters naturally synthesised by some bacteria and archaea. PHA have high industrial value as bioplastics for packaging and biomedical applications. However, their broader use is hindered by high production costs and uncontrolled variation of polymer properties. The extreme halophile Haloferax mediterranei shows bioprocess advantages that can be exploited for the low cost production of the PHA copolymer Poly(3-hydroxbutyrate-co-3-hydroxyvalterate) (PHBV). The focus of this thesis is to identify process variables responsible for the uncontrolled variation of PHA properties in order to progress towards the robust production of PHBV using H. mediterranei. The outcome of the investigation is a novel cultivation strategy for the reliable synthesis of PHBV copolymers with controlled composition and microstructure showing minor differences in material characteristics. Initially, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and for the first time, nitrate. The nitrogen source and concentration influenced PHBV accumulation and variations in polymer composition were observed with ammonium, highlighting the importance of the control of cultivation conditions. Volatile fatty acids (VFA) were found to be a more direct approach to determine PHBV composition and for the first time were used as substrates in H. mediterranei cultures. When the cells were grown in C4:0/C5:0 mixtures, the 3HV fraction in the PHBV was proportional to the percentage of C5:0 in the feed mixture, allowing the synthesis of copolymers with a predefined composition ranging from pure PHB to pure PHV. The cultivation strategy proved effective for the synthesis of HV rich PHBV, which is not easily obtained due to the 3HV precursor toxicity. The polymer microstructure was controlled using different feeding strategies: co-feeding generated random copolymers, while sequential feeding created block and blend copolymers. The synthesis of block copolymers is of interest because the materials show enhanced yield strength and mechanical strength, making such materials more suitable for commodity uses. Bespoke random, block, and blend copolymers with 0−100 mol% 3HV were synthesized and their thermal and mechanical properties studied. Lastly, high temperature cultivation and several surfactants were tested to enhance the production of bespoke PHBV from VFA. PHBV productivity and accumulation was greatly improved in a fed-batch bioreactor fermentation at 37°C with Tween-80 and the maximum PHBV content 58.9% was obtained. The polymers from shake-flasks and from bioreactors showed minor variations in their material properties, demonstrating the scalability and the robustness of the process developed. Further understanding of the different process variables affecting polymer synthesis and composition was gained in this thesis. It is now possible to produce PHBV with controllable composition, microstructure and minor differences in material characteristics. The novel and robust production strategy developed address the bioprocess challenge of minimising the uncontrolled variation of polymer characteristics that is currently hindering the wider use of PHA hence allowing the production of high quality polymers for commodity goods, packaging and biomedical applications.

I Sverige förekommer strikta krav på fosforrening av avloppsvatten och detta har bidragit till att kemisk fällning har kommit att dominera som reningsmetod för fosfor vid svenska avloppsreningsverk. Fällningskemikalier är dyrt för reningsverken att köpa in och ger negativ påverkan på miljön vid tillverkning och transport. Strängare reningskrav har ökat behovet av nya reningsmetoder som på ett effektivt och miljövänligt sätt kan rena avloppsvatten från näringsämnen utan att kostnaderna för reningen blir för stora. Biologisk fosforavskiljning (bio-P) utnyttjar mikroorganismer som naturligt kan ackumulera fosfor i sina celler. En kritisk faktor för en väl fungerande biologisk fosforavskiljning är tillgången till kolkälla i form av flyktiga fettsyror (VFA). Kommunalt avloppsvatten innehåller vanligen för lite VFA för att tillgodose bio-P-processens behov. Den totala tillgången på organiskt material i avloppsvattnet är dock ofta stor och möjligheten finns därmed att genom biologisk slamhydrolys internt producera VFA. Vid biologisk hydrolys av slam utnyttjas mikroorganismers naturliga förmåga att under anaeroba förhållanden bryta ned organiskt material till mer lättomsättliga föreningar. Duvbackens avloppsreningsverk i Gävle tillämpar biologisk fosforavskiljning och producerar VFA till bio-P-processen genom primärslamshydrolys och sidoströmshydrolys av returslam. I detta examensarbete har hydrolysprocesserna vid reningsverket utvärderats med avseende på processernas förmåga att producera VFA till det biologiska reningssteget. Utvärderingen gjordes genom att mäta förändringen i halten organiskt material över hydrolysbassängerna samt genom att bedöma effektiviteten i bio-P-processen i fullskala och vid labskaleförsök. Möjligheten att stänga av primärslamshydrolysen vid verket undersöktes. Ett försök till att optimera driften av sidoströmshydrolysen gjordes genom att utföra hydrolysförsök i laboratorieskala. Resultaten indikerade att produktionen av VFA i primärslamshydrolysen var begränsad och att processen därmed bör kunna stängas av. Innan detta görs bör dock kompletterande mätningar av halten löst COD över primärslamshydrolysen utföras. Vid samtliga mättillfällen uppmättes en betydande ökning i halten organiskt material över sidoströmshydrolysen. Baserat på detta och den i examensarbetet konstaterade effektiviteten i bio-P-processen drogs slutsatsen att sidoströmshydrolysprocessen vid Duvbackens reningsverk var välfungerande. Hydrolysförsöken pekade på att ett större utbyte av VFA skulle erhållas om en kortare uppehållstid än den nuvarande användes i sidoströmshydrolysen. Detta bör vidare undersökas genom fullskaleförsök vid verket. / The strong regulations concerning phosphorus removal from wastewater in Sweden has resulted in chemical precipitation being the most common treatment method for phosphorus at Swedish wastewater treatment plants (WWTP). Precipitation chemicals are expensive and have a negative environmental impact when produced and transported. More stringent wastewater treatment requirements have increased the need of new, eco-friendly treatment methods that effectively can remove nutrients from wastewater without being too expensive. Enhanced biological phosphorus removal (EBPR) utilizes microorganisms that have the natural capability of accumulating phosphorus in their cells. A critical factor for a well-functioning EBPR-process is the availability of carbon source in form of volatile fatty acids (VFA). Municipal wastewater normally contains too small amounts of VFA to satisfy the need of the EBPR-process. The total amount of organic matter in the wastewater is on the other hand large and the possibility consequently exists to internally produce VFA through sludge hydrolysis. Biological sludge hydrolysis takes advantage of the natural capability of microorganisms to degrade complex organic compounds into easily degradable organics. Duvbacken WWTP in Gävle uses EBRP for phosphorus removal and produces carbon source through hydrolysis of primary sludge and sidestream hydrolysis of return sludge. In this master thesis the hydrolysis processes at the WWTP was evaluated with regard to the capacity of the processes to produce VFA to the biological treatment step. The evaluation was performed by measuring the change in organic material content over the hydrolysis basins and by estimating the effectiveness of the EBPR-process in full scale and by laboratory experiments. The possibility to turn off the primary sludge hydrolysis process was examined. An attempt to optimize the operation of the sidestream hydrolysis process was made by conducting hydrolysis experiments in laboratory scale. The results indicated that the production of VFA by primary sludge hydrolysis was limited and that it thus would be possible to turn off the process. Before this is done complementary measurements of COD levels over the primary hydrolysis basin should be performed. At all times considerable increments in COD content was measured over the sidestream hydrolysis basin. Based on this and the in the thesis confirmed effectiveness of the EBRP-process the conclusion was drawn that the sidestream hydrolysis of return sludge at Duvbacken WWTP was well functioning. The hydrolysis experiments indicated that a larger VFA yield would be obtained if a shorter retention time than the present was used in the sidestream hydrolysis process. This should further be investigated by experiments in full scale at the WWTP.

Ce travail de thèse a eu pour objectif principal l'étude thermodynamique des changements métaboliques dans l'acidogénèse. L'acidogénèse est un procédé anaérobie à double intérêt qui en traitant des résidus organiques, permet de produire des composés chimiques comme l'hydrogène, l'éthanol et les acides organiques. Par conséquent, l'acidogénèse se place comme un procédé biotechnologique dans le concept de bioraffinerie. En outre, ce processus n'a pas besoin de conditions stériles d'opération et fonctionne sur une large gamme de pH. Ces changements métaboliques sont dépendants des modifications dans les conditions opératoires. Afin d'étudier ces changements métaboliques, des expériences basées sur des modifications du ciel gazeux du réacteur par introduction d'azote et sur des changements du pH, ont été menées. Un des résultats les plus intéressants a été l'augmentation du rendement de production d'hydrogène de 1 à 3,2 molH2/molglucose à pH 4,5 et débit de N2 de 58,4 L/d. Ce rendement est proche de la valeur théorique (4 molH2/molglucose). L'étude thermodynamique a permis d'expliquer les mécanismes métaboliques concernant l'hydrogène, dont la production importante, représentée par le rendement de 3,2 molH2/molglucose, est due à la réaction inverse H2/NAD+, qui est thermodynamiquement faisable à faibles pressions partielles d'hydrogène (par exemple 0,02 bar). En outre, les bas rendements en hydrogène ont été expliqués par l'action consommatrice d'hydrogène par la réaction d'homoacetogénèse. Cependant, le modèle n'a pas été capable d'expliquer les changements métaboliques de l'acétate, du butyrate et de l'éthanol lors de la fermentation acidogénique du glucose. / This thesis deals with thermodynamic based modelling of metabolic shifts during acidogenic fermentation. Acidogenic fermentation is an anaerobic process of double purpose: while treating organic residues, it produces chemical compounds, such as hydrogen, ethanol and organic acids. Therefore, acidogenic fermentation arises as an attractive biotechnology process towards the biorefinery concept. Moreover, this process does not need sterile operating conditions and works under a wide range of pH.Changes of operating conditions produce metabolic shifts, inducing variability on acidogenic product yields. In order to study these metabolic shifts, an experiment design was based on reactor headspace N2-flushing (gas phase) and pH step changes (liquid phase). A major result was the hydrogen yield increase from 1 to 3.2 (molH2/molglucose) at pH 4.5 and N2-flushing of 58.4 L/d. This yield is close to the theoretical acidogenic value (4 molH2/molglucose).The thermodynamic model, based on the assumption that acidogenic fermentation is characterised by limited energy available for biological process, allowed to explain the mechanisms that govern hydrogen metabolic shifts, showing that the synthesis of extra hydrogen, i.e. yield of 3.2 (molH2/molglucose), was due to reverse H2/NAD+ redox reaction, which is thermodynamically feasible at low hydrogen partial pressures (e.g. 0.02 bar). Moreover, low hydrogen yields were explained by the action of homoacetogenesis hydrogen consuming reaction. However, the model was not capable to explain the metabolic shifts of acetate, butyrate and ethanol on acidogenic glucose fermentation.

Biodiversity is a key component for life on Earth since it contributes to clean water, fresh air and food security. Today, fatty fish farmed in aquaculture is the main Omega 3 source consumed by humans, including the essential fatty acid docosahexaenoic acid (DHA). DHA origin from plants and accumulate in fish via the marine food web. Therefore, DHA in the form of fish oil is often added to fish feed used in aquaculture. This process is dependent on fossil energy and marine raw materials, which infer increased global warming, damage to ecosystem and ultimately loss of biodiversity. In order to reduce the environmental impact, the essential fatty acid could instead be derived from the marine primary producer of DHA: microalgae. In this thesis, a life cycle approach was used to assess global warming, use of fossil fuels and Ecosystem damage when DHA is produced by the microalgae Crypthecodinium Cohnii. The environmental impact was modelled using SimaPro 9 and assessed with CML-IA and ReCiPe Endpoint. In this model, volatile fatty acids derived from dark fermentation of food waste was used as feedstock to the algae. The studied systems consisted of two parallel scenarios, one conventional food waste-to-biogas with DHA from fish oil and one conceptual food waste-to-DHA with DHA from algae oil. The aim was to evaluate the future potential of DHA produced from algae, by assessing and comparing environmental impact to DHA produced from Peruvian anchovy. For every ton DHA produced by microalgae the assessed impact was -1.9E+02 tonCO2e, -1.9 TJ and 9.7E-04 species.yr. DHA produced by microalgae using VFA from food waste was shown to mitigate global warming and reduce use of fossil fuels. The most important conclusion show that DHA from algae infer 37% lower biodiversity loss in comparison to DHA from Peruvian anchovy. Thus, DHA from microalgae could reduce dependency on marine raw material and decrease biodiversity loss. / Biodiversitet är en nyckelkomponent för liv på jorden eftersom det bidrar till rent vatten, frisk luft och säker livsmedelsproduktion. Idag är fet fisk odlad i vattenbruk den viktigaste källan till Omega 3 som konsumeras av människor, inklusive den essentiella fettsyran dokosahexaensyra (DHA). Då DHA härstammar från växter och ackumuleras i fisk via den marina näringskedjan, tillsätts DHA ofta till fiskfoder i form av fiskolja. Denna process är beroende av fossil energi och marina råmaterial, som leder till ökad global uppvärmning, skadar naturliga ekosystem och orsakar förlust av biologisk mångfald. För att minska miljöpåverkan skulle den essentiella fettsyran istället kunna produceras från den marina primärproducenten av DHA: mikroalger. I detta examensarbete användes livscykelanalys för att utvärdera miljöpåverkan med avseende på global uppvärmning, användning av fossila bränslen och påverkan på biodiversitet, då DHA produceras av mikroalgen Crypthecodinium Cohnii. Flyktiga fettsyror, VFA, som bildas vid mörk fermentering av matavfall användes som råmaterial till algerna. De studerade systemen bestod av två parallella scenarier, en konventionell matavfall-till-biogas med DHA från fiskolja och en konceptuell matavfall-till-DHA med DHA från algolja. Systemet modellerades i SimaPro 9 och miljöpåverkan beräknades med CML-IA och ReCiPe Endpoint. Syftet var att utvärdera DHA som produceras från alger genom att beräkna miljöpåverkan och jämföra med DHA producerad från peruansk ansjovis. För varje ton DHA producerat av mikroalger var påverkan -1.9E+02 tonCO2e, -1.9 TJ och 9.7E-04 arter per år. DHA producerad av mikroalger där VFA från matavfall använts som näring, visade sig minska den globala uppvärmningen, reducera användningen av fossila bränslen och innebar 37% lägre förlust av biologisk mångfald jämfört med DHA producerad från peruansk ansjovis. Denna studie visade därmed att DHA från mikroalger kunde minska beroendet av marina råmaterial och minska förlusten av biologisk mångfald.