This thesis investigates a newly developed and patented technology for its ability to inactivate spore- forming bacteria and non-spore-forming microorganisms. This new technology “Pressure Assisted Thermal Sterilization©” (PATS) is based on the theory of the thermal expansion of liquids. The efficiency of inactivating spore-forming and non-spore-forming microorganisms by PATS was compared with the thermal treatment alone. A combination treatment consisting of high pressure processing and gaseous carbon dioxide was also investigated for its ability to inactivate bacterial spores in model and real food matrices. The structural damage caused by treatments to the spores and non-spore-forming bacteria was assessed by scanning electron microscopy. Geobacillus stearothermophilus spores suspended in Milli-Q water, UHT milk and pumpkin soup, treated by PATS were found to have significantly lower decimal reduction times (D values) compared with the thermal treatment alone. Spores suspended in UHT milk were more heat resistant compared with those in Milli-Q water and pumpkin soup. Bacillus cereus spores suspended in Milli-Q water and pumpkin soup treated with PATS were more effectively inactivated compared with spores treated by the thermal treatment alone. Clostridium botulinum spores in saline buffer subjected to PATS treatment were inactivated more effectively compared with the thermal treatment alone. Overall, the results show that PATS was a better processing technique for inactivation of bacterial spores compared with thermal treatment alone. However, PATS had no added benefit in inactivating the non-spore-forming bacteria Escherichia coli and Saccharomyces cerevisiae cells compared with the thermal treatment. A shelf life study showed that B. cereus spores in pumpkin soup retained a low spore count (<5 LogCFU/mL) for approximately 40 days in 30oC storage after treatment with PATS. No additional degradation of colour pigments of pumpkin soup and model pumpkin juice was observed following PATS compared with the thermal treatment. Spore-forming microorganisms can be resistant to pressure treatment alone, which limits the application of high pressure processing (HPP). Therefore, a combination approach was investigated. The mechanism of inactivating spores by combining HPP with other treatments is that the pressure assists in spore germination. Then a secondary treatment (thermal or CO2 gas) can be used to inactivate the germinated spores. A combined application of HPP and a consecutive CO2 treatment was investigated for the efficiency of spore inactivation. Results showed that HPP (200 MPa for 30 min) followed by a CO2 treatment inactivated Bacillus subtilis 168 in nutrient broth, tomato juice and liquid whole egg by 2.5, 1.0 and 1.5 LogCFU/mL respectively. These results indicated that this technique is inadequate for practical use. Scanning electron micrographs showed that pressure processing of B. subtilis 168 and B. subtilis natto spores resulted in deformation of the spore structure. This structural deformation of spores may have been due to water absorption during HPP and subsequent release upon decompression. PATS treated G. stearothermophilus and B. cereus spores were more severely damaged compared with the same spores which underwent thermal treatment alone. However, the extent to which E. coli and S. cerevisiae cells were damaged by both PATS and thermal treatment was similar.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with bad prognosis, which predominates in males with advanced age. However, studies of the endocannabinoid system and how it affects tumour behaviour provides the basis for designing innovative therapeutic strategies that could open new opportunities for treatment of patient with MCL. It has earlier been shown that the cannabinoid receptor ligand (R)-(+)-methanandamide (R-MA) induce cell death in MCL by accumulation of ceramide. Ceramide has a pro-apoptotic effect on the cell but could be metabolized by the enzymes glucosylceramide synthase (GCS) and sphingosine kinase 1 (SphK1) to molecules with pro-proliferative effect. Therefore, treatments with R-MA on Jeko-1 MCL cell line were performed in this study to determine interference in the proliferative behaviour as well as in the gene expression of the enzymes GCS and SphK1. In addition, treatments with chemotherapeutic substances, such as doxorubicin or cytarabine (Ara-C), and combinations of R-MA and chemotherapeutic substance, were performed for the same reason. Results showed that the proliferation behaviour of Jeko cells remained unaffected when treated with R-MA, in contrast to the decreased proliferative effects shown when treated with cytostatics or combinations of R-MA and cytostatics. Furthermore, a tendency for up-regulation of GCS and SphK1 expression was recognized when cells were treated with cytostatics or combination of cytostatics and R-MA, in contrast to cells treated with R-MA alone. Although, R-MA alone had a tendency for a small down-regulation of GCS expression, it contributed to a potential elevation of GCS expression when combined with Ara-C or doxorubicin. It is believed that the effect from upregulated levels of the metabolizing enzymes GCS and SphK1 is balanced by, earlier observed, up-regulations of the ceramide synthesis enzymes.

The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells. Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation. Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics. In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity. Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well. / <p>QC 20101129</p>

The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium. Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex. / QC 20101125

pH is a key parameter in the optimization of animal cell processes, and has be linked to specific patterns of consumption and production of extracellular metabolites. However, the effect of extracellular pH on intracellular metabolism has not been fully elucidated. Metabolic flux analysis is a mathematical method that can be used to generate the intracellular flux distributions in cells, e.g. as a function of some environmental parameter. In this work, the overall objective was to develop a culture system and experimental protocol for cultivation of CHO cells, which can be used to generate data for analysis of the relationship between extracellular pH and intracellular fluxes in CHO cells by metabolic flux analysis. First, shake-flask culture of an IgG-producing cell line was performed to select an academic and chemically-defined medium with known composition. This was followed by subsequent adaptation of the cells. It was found that the originally selected medium had to be supplemented with a commercial medium to produce acceptable growth and viability. Shake-flask culture was also performed to evaluate the effect of the biological buffer HEPES on cell growth and viability, and the pH-stability during culture. HEPES-concentrations in the investigated range (7.5-45 mM) did not show an apparent effect on cell growth or viability. The higher concentrations gave slightly better buffering capacity at inoculation, however were not sufficient to keep pH stable during culture. As a result, the idea of using shake flask culture and similar techniques for cultivation of cells at various pH set-points was dismissed. Instead, a culture system and protocol based on a 100 mL Spinner flask with pH-regulation was custom-designed for the project. Features of the final design included continuous monitoring of pH and DO, stable temperature at 37 °C, adjustable agitation rate, as well as the option to incorporate inflow of air, O2 and CO2. In addition, the possibility to disconnect the flask unit to perform medium exchange and sample collection away from the reactor site (i.e. in a laminar flow workbench) was integrated into the design and protocol. The system was demonstrated for pseudo-perfusion culture with the adapted IgG-producing cell line at pH 7.0 during 24 days. Optimized regulation settings were identified. It was shown that the system could support viable cell densities of up to 11 MVC/mL and high viability (&gt; 90 %). During the final phase of culture, stable growth, at specific growth rates of approximately 0.7 Day-1, was achieved. The specific rates of consumption and production of the key metabolites glucose, glutamine, lactate and NH4+, as well as 20 amino acids were analyzed. A majority of the rates were in accordance with CHO cell metabolism. The expected consumption of a majority of the essential amino acids and main carbon sources glucose and glutamine were confirmed, as well as the associated production of by-products lactate and NH4+. The system and protocol developed in this work can be used in future experiments to generate data describing metabolic profiles as a function of various pH-set points. This data may then be used in metabolic flux analysis to further elucidate the metabolism behind pH effects in CHO cells. / pH är en viktig parameter i optimeringen av animalcellsprocesser och har sammankopplats med specifika konsumtions- och produktionsmönster rörande extracellulära metaboliter. Det extracellulära pH-värdets effekt på den intracellulära metabolismen är dock inte fullt klarlagd. Metabolisk flux analys är en matematisk metod som kan användas för att generera intracellulära fluxfördelningar i celler, exempelvis som en funktion av någon yttre parameter. Det övergripande målet i detta arbete var att utveckla ett odlingssystem och experimentellt protokoll för odling av CHO-celler som kan användas för att generera data för metabolisk flux analys där målet är att studera effekten av pH på den intracellulära cellmetabolismen. En IgG-producerande CHO-cellslinje odlades först i skakkolv för att välja ut ett akademiskt kemiskt definierat medium med känd sammansättning. Därefter följde försök att anpassa cellerna till det valda mediet. Det visade sig att ett kommersiellt medium behövde tillsättas för att ge godtagbar tillväxt och viabilitet. Effekten av den biologiska bufferten HEPES på cellernas tillväxt och viabilitet, samt pH-stabiliteten under odling, undersöktes också genom odling i skakkolv. HEPES-koncentrationer i det undersökta intervallet (7.5 – 45 mM) hade ingen större effekt på tillväxt och viabilitet. För de högre koncentrationerna var buffertkapaciteten något bättre precis vid inokulering. Dessa koncentrationer var dock ej tillräckliga för att ge stabilt pH under odlingen. Baserat på dessa resultat övergavs tanken på att använda skakkolvsodling för att odla celler vid olika pH-värden. Ett odlingssystem och ett protokoll baserat på en 100 mL Spinnerflaska med pH-reglering specialdesignades istället för projektet. I det färdiga systemet fanns lösningar för kontinuerlig övervakning av pH och DO, stabil temperatur vid 37 °C, justerbar omrörningshastighet, samt valmöjligheten att flöda in luft, O2 och CO2. Dessutom infördes möjligheten att koppla loss flaskenheten från reglersystemet för byte av medium och provtagning. För att demonstrera systemet genomfördes en odling med den anpassade IgG-producerande cellinjen enligt principen för pseudo-perfusion vid pH 7.0. Odlingen pågick under 24 dagar och optimerade reglerinställningar identifierades. Det visades att systemet kunde understödja cellkoncentrationer upp till 11 miljoner celler per milliliter, samt hög viabilitet (&gt; 90 %). Under den senare delen av odlingen uppnåddes stabil tillväxt, vid specifika tillväxthastigheter omkring 0.7 per dygn. Den specifika konsumtions- och produktionshastigheten för metaboliterna glukos, glutamin, laktat och NH4+, samt 20 aminosyror analyserades. Majoriteten av hastigheterna stämde överens med typisk CHO-cellsmetabolism. Den förväntade konsumtionen av majoriteten av de essentiella aminosyrorna och huvudsakliga kolkällorna glukos och glutamin konfirmerades, såväl som den associerade produktionen av bi-produkterna laktat och NH4+. Odlingssystemet och det experimentella protokollet som utvecklades i detta arbete kan användas i framtida experiment för att generera data som beskriver metaboliska profiler som funktion av extracellulärt pH. Dessa data kan sedan användas i metabolisk flux analys för att dra slutsatser om pH-effekter i CHO-celler.

<p>Microbes play key roles in processes that shape the world around us having direct impact in crop  production,  food  safety,  digestion,  and  overall  health.  Developing  tools  to  monitor  their activity in-situ is the key towards better understanding the true impact of microbial activity in these processes and, eventually, harnessing their potential. Many conventional techniques for microbial activity assessment require sample collection, expensive benchtop equipment, skilled technicians, and   destructive   sample   processing   which   makes   their   adaptation   for   in-situ   monitoring cumbersome. The need for technologies for in-situ monitoring has led to the development of many sensordesigns,  capable  of  detecting  single  strains  of  bacteria  to  low  limits  of  detection  (LOD). These designs, however, are limited to their complex manufacturing procedures, cost, and delicacy which makes them difficult to implement outside of a laboratory setting into harsh environments.</p> <p>In  the  last  25  years,  impedimetric  sensing  methods  have  been used  as  powerful  analytical tools  to  characterize  the  degradation  and  dissolution  of  polymers.  Known  for  their  robustness, these techniqueswere mainly used for characterizing polymer’s properties as corrosion-protective layers on metals. At the time, someresearchers pondered onthe potential use of this technique for biosensing  applications.In  this  thesis,  the  ability  of  monitoring  microbial  activity  in-situ  was explored by  integratingdifferent  bioresponsive  polymers  with  low-cost electronic  impedimetricplatformsand assessing their degradation kinetics in response to microbes</p> <p>This  novel  use  of  impedimetric  sensing  methods  and  approach  towards  microbial  activity sensing was systematically studied in different  areas including  agriculture, food packaging, and healthcare.  Microbes,  the  good,  the  bad,  and  the  ugly,  were  studied  within  their  ecosystems  to demonstrate  the  ability  of  using  the  described  systems  in  in-situ  monitoring.  In  agriculture, polymer  degradation  was  successfully  correlated  to  the  concentration  of  decomposing  bacteria directly in soil. In food packaging, spoilage of chicken samples was successfully detected within their package through a non-reversible system. In healthcare, a wireless and electronic-free wound monitoring  system  capable of  detecting  early  onset  of  infection  while  delivering  therapeutics without the need of external actuation was achieved. Further developments of this technology will present the key towards monitoring microbial activity in-situ in a large scale, providing solutions to  humanity’s  toughest  upcoming  challenges  including  food  production,  food  safety,  and healthcare.</p>

Single cell proteins such as that of edible filamentous fungal biomass are considered as a promising sustainable source of animal feed supplementation. Filamentous fungi can be cultivated on different organic substrates including volatile fatty acids (VFAs) such as acetic, propionic, and butyric acids. These VFAs can be generated through the famous waste valorisation approach of anaerobic digestion (AD) as intermediate metabolites. This project investigates a sustainable approach for the production of animal feed supplementation through cultivation of fungal biomass on waste derived VFAs along with the in vitro analysis of fungal biomass digestibility as ruminant feed. In this regard, optimum conditions for the production of Aspergillus oryzae biomass on different VFAs effluents derived from anaerobic digestion process of food waste plus chicken manure (FWCKM) and potato protein liquor (PPL) at different pH, nitrogen sources, and feed mixture was studied. Accordingly, analyses showed that PPL has the highest biomass yield with 0.4 (g biomass/g consumed VFAs) based on the volatile solids (VS) by adjusting pH to 6.2. Furthermore, the digestibility of the produced fungal biomass is analysed by using three different in vitro digestion methods including Tilley and Terry (TT) method, Gas Production Method (GPM), and Nylon Bag Method (NBM) and the results are compared with the conventional feed (silage and rapeseed meal). Results obtained from different digestibility methods illustrate that different A. oryzae fungal biomass had approximately 10-15 % higher dry matter digestibility fraction compared to silage and rapeseed meal (reference feeds). Hence, these results revealed that A. oryzae fungal biomass can grow on VFAs effluents and produce protein-rich fungal biomass while this biomass has better digestibility compared to conventional feeds and confirmed the initial hypothesis of the study.

In conventional wastewater treatment plants, methanol, ethanol, and acetate are used as carbon source for the denitrification process in the biological nutrient removal. However, growing concern regarding economical costs and carbon footprints from the fossil-based production of these chemicals have forced the companies to look for other alternatives. VFAs have shown a great potential in replacing the conventionally used carbon sources. If implemented this will result in lower chemical cost and a drastic decrease in carbon footprint as well as striving WWTPs towards sustainable development. In this work denitrification has been analysed using different variations of VFAs such as fermented potato protein liquor, food waste and chicken manure VFA. This was done using a basic laboratory setup of a denitrification reactor which used basic stirring agitation and nitrogen purging to ensure anoxic conditions. Nutrients and excess sCOD were added to ensure the highest denitrification rates. The denitrifying biomass was collected at Gryaab AB in the form of k1-carriers making this process a MBBR. The most influential characteristic of the VFAs is the distribution of the acids in the VFA effluent. Butyric acid along with caproic acid showed the best potential for efficient denitrification. The possibility of concentration of VFA effluent showed a high potential when using a nanofiltration system. A C/N ratio of 4.5 conventionally used when methanol is added showed to be the most optimal condition for VFA addition. The combination of VFAs together with conventional used carbon sources showed the best potential in denitrification efficiency proving to be as good or even better than pure synthetic ones. VFAs effluents showed the best potential in removing the intermediate nitrite from the wastewater at high rates. Overall, VFAs shown a great potential for replacing conventionally used carbon sources, demonstrating the potential of substitution, which if implemented will result in lower carbon footprint and a strive towards sustainable development.

Under de senaste 30 åren så har antalet massa- och pappersbruk minskat medan den totala produktionen av massa och papper har ökat. En högre produktion per bruk tillsammans med en hårdare miljölagstiftning, sätter ett högre tryck på vattenreningen på varje bruk. I flera massa- och pappersbruk så är vattenreningen en av faktorerna som begränsar produktionen av massa och papper. Dessutom så renas oftast restvattnet från massa- och pappersbruken med aerob rening som kräver mycket elektricitet och som producerar slam. För att minska kostnaden av vattenreningen och för att öka kapaciteten, så kan en anaerob vattenrening användas före den aeroba reningen. Fördelar med att ha en anaerob rening före den aeroba reningen är att den minskar belastningen av organiskt material på den aeroba reningen, värdefull biogas produceras, anaerob rening producerar mindre slam och kräver mindre elektricitet. Däremot så innehåller restvattnet från massa- och pappersbruk en hög halt av sulfat, vilket har orsakat en instabil biogasproduktion på flera existerande anläggningar. En potentiell lösning på detta är att använda en ytterligare anaerob reaktor före den biogasproducerande reaktorn, med syftet att reducera sulfat. Syftet med detta projekt var att optimera en anaerob två-stegs process för att få en effektiv biogasproduktion och för en stabil process. I projektet har två upp-flöde anaerob packad bädd (UAPB) reaktorer använts. Den första reaktorn var anrikad med sulfatreducerande bakterier före starten av projektet och den andra reaktorn innehöll metanogener. Processvatten från ett massa- och pappersbruk som använder barrträd som råmaterial och gör termomekanisk massa, användes i projektet. Den anaeroba två-stegs processen optimerades genom att minska retentionstiden i båda reaktorerna; genom att undersöka alternativ för att minska sulfidkoncentrationen i inflödet till biogasreaktorn; och genom att minska tillsatsen av näringsämnen till processvattnet. Stabiliteten av två-stegsprocessen mättes genom att analysera sulfat, sulfid och COD innehållet med spektrofotometri; genom att analysera pH; genom att analysera innehållet av metan i biogasen med gaskromatografi; och genom att analysera flyktiga fettsyror i utflödet med högtrycksvätskekromatografi. Resultaten visade att processen var stabil när retentionstiden för den sulfatreducerande reaktorn och biogasreaktorn var 0.3 dagar respektive 1.5 dagar. Effektiviteten av borttagningen av sulfat och COD i den sulfatreducerande reaktorn var 82% respektive 31%. Innehållet av metan i biogasen i den sulfatreducerande reaktorn var 53% i genomsnitt. I biogasreaktorn var borttagningen av COD 32% och metanhalten i biogasen var 31% i genomsnitt. Den totala borttagningen av COD för tvåstegsprocessen var 61%, när reaktorerna var kopplade via en uppsamlingsflaska. Biometanpotentialen i processvattnet och i utflödet från den sulfatreducerande reaktorn var 147 NmL CH4/g VS respektive 47 NmL CH4/g VS. Slutsatsen av projektet är att processen var stabil när retentionstiden för den sulfatreducerande reaktorn och biogasreaktorn var 0.3 dagar respektive 1.5 dagar, motiverat med stabiliteten av borttagning av sulfat och COD. Däremot var metanpotentialen för utflödet från den sulfatreducerande reaktorn bara 32% jämfört med processvattnet och nedbrytbarheten av COD var lägre i utflödet än i processvattnet. Eftersom den sulfatreducerande reaktorn var stabil vid de testade förhållandena så skulle det vara intressant att testa en enstegsprocess i en UAPB reaktorpilot i framtiden. Det skulle även vara intressant att minska retentionstiden i biogasreaktorn för att se om metanhalten i biogasen ökar. / In the past 30 years, the number of P&amp;P mills have decreased while the total production of pulp and paper have increased. A higher production per mill together with a stringent environmental legislation puts a high pressure on the wastewater treatment of P&amp;P mills. In several P&amp;P mills the wastewater treatment is one of the factors limiting the production of pulp and paper. In addition, the wastewater is usually treated with aerobic treatment that consumes a high amount of electricity and produces sludge. To decrease the cost of the wastewater treatment and to increase the treatment efficiency, an anaerobic treatment can be used before the aerobic treatment. Advantages of having an anaerobic treatment before the aerobic treatment is that it reduces the organic loading of the aerobic treatment, valuable biogas is produced, anaerobic treatment produce less sludge and requires less electricity. However, the wastewater of P&amp;P mills is rich in sulphate, which have resulted in an unstable biogas production. A potential solution for this to have another anaerobic reactor before the biogas producing reactor, with the purpose to remove sulphate. The aim of this project was to optimize a two-step anaerobic treatment for an effective biogas production and for a stable process. In the project, two UAPB reactors were used. The first bioreactor (hereafter pretreatment reactor) was enriched with sulphate reducing bacteria before the start of the project while the second reactor contained methanogens (hereafter biogas reactor). Wastewater from a P&amp;P mill using the TMP process and soft wood as raw material was used in this project. The anaerobic two-step process was optimized by decreasing the HRT of both reactors, by investing alternatives to decrease the sulphide in the influent to the biogas reactor and by decreasing the addition of nutrients to the process water. The stability of the two-step process was measured by analysing the sulphate, sulphide and COD content spectrophotometrically; by analysing pH; by analysing the methane content in the headspace of the reactors with GC and by analysing the VFA content in the effluents using HPLC. The results showed that the process was stable when the HRT of the pretreatment reactor and biogas reactor was 0.3 days and 1.5 days, respectively. The removal efficiency of sulphate and COD in the pretreatment reactor was 82% and 31%, respectively. The methane content in the biogas of the PT reactor was 53% in average. The COD removal efficiency of the biogas reactor was 32% and there was in average 31% methane in the biogas. The average total COD removal efficiency of the two-step process was 61% when the reactors were coupled via a collection tank. The biomethane potential of the process water and the effluent of the pretreatment reactor was 147 NmL CH4/g VS and 47 NmL CH4/g VS, respectively. The process was stable when the HRT of the pretreatment reactor and biogas reactor was 0.3 days and 1.5 days, respectively, considering the stability of the sulphate and COD removal efficiency. However, the BMP of the effluent of the PT reactor was only 32% of the BMP of the process water and the degradability of the COD was lower in the effluent. Since the PT reactor exhibited stability at the tested conditions, it would be interesting to try it as a one-step process in a pilot UAPB reactor in the future. In the future, research is needed to investigate if the methane content in the headspace of the biogas reactor can be increased by decreasing the HRT of the reactor.

<p>The development of low-cost, and reliable platforms for on-site detection of pathogenic agents, and toxic environmental traces is still a critical need for real-time monitoring of potential environmental pollution and imminent outbreaks. The biosensors market is projected to attain 31.5 billion by 2024. In this landscape, colorimetric and electrochemical devices continue to have significant relevance, with paper-based platforms leading the point-of-care (POC) segment for pathogen detection and environmental monitoring.</p> <p>Despite the true potential of biosensors in general, they have witnessed a slow rate in commercialization, mainly due to cost restrictions, and concerns related to their reliability and repeatability once scaled-up. This research evaluates the implementation of printing techniques as a strong approach for the fabrication of paper-based and flexible electrochemical biosensors. The results obtained demonstrated the ability to control and predict the variables affecting the sensing performance, achieving high precision of the printing parameters, and allowing optimization, and iterations since very early stages of prototype development.</p> <p>Besides the novel fabrication approach, this work introduces the use of truncated aptameric DNA sequences for whole cell detection of E. coli O157:H7 and heavy metals (Hg2+ and As3+), providing evidence of high stability and robustness under harsh conditions. Results obtained demonstrate their equal or even superior performance when compared to antibodies.</p> <p>We established the use of aptamer-functionalized multilayered label particles (PEI-grafted gold decorated polystyrene) with high stability as label particles. These particles address the well known drawback of non-selective aggregation typical of traditional naked Gold nanoparticles. The outstanding stability of these multilayered labels was demonstrated when used in an enhanced version of the lateral flow assay for detection of E. coli O157:H7 (state of the art for paper-based colorimetric detection of whole cell bacteria), and in a multiplexed paper-based microfluidic device for dual detection of Mercury and Arsenic. This work sets the foundation of the development of a next generation of health care and environmental monitoring devices that are portable, sensitive, quantitative, and can reliably detect multiple targets with one single test.</p>