A major source of CO2 emissions is the flaring of steel mill gas. This work demonstrated the enrichment of carboxydotrophic bacteria for converting steel mill gas into volatile fatty acids and H2, via gas fermentation. Several combinations of pure and mixed anaerobic cultures were used as inoculum in 0.5-L reactors, operated at 30 and 60 °C. The process was then scaled up in a 4-L membrane bioreactor, operated for 20 days, at 48 °C. The results showed that the enriched microbiomes can oxidize CO completely to produce H2/H+ which is subsequently used to fix the CO2. At 30 °C, a mixture of acetate, isobutyrate and propionate was obtained while H2 and acetate were the main products at 60 °C. The highest CO conversion and H2 production rate observed in the membrane bioreactor were 29 and 28 mL/LR/h, respectively. The taxonomic diversity of the bacterial community increased and the dominant species was Pseudomonas.

MultiBio project aimed to establish and demonstrate a novel multipurpose biorefinery cascade concept, producing three renewable biobased products: 1) biohydrogen, 2) biopolymers and 3) protein rich meal ingredients for fish farming. The cascade concept exploits the ability of a bacterium (Caldicellulosiruptor saccharolyticus) to transform nutrients present in low-value waste process waters of the pulp and paper industry, to high-value products hydrogen gas, organic acids and microbial biomass. The organic acid rich effluent will then be managed in an open culture microbial process used to achieve discharge water quality objectives and to produce polyhydroxyalkanoate (PHA) biopolymers. Moreover, since C. saccharolyticus protein content is more than 63% of cell dry weight, their potential in formulation of fish feed was evaluated. 

A fiber sludge containing, CTMP residual stream was found to be a possible feedstock for the MultiBio process concept. Due to safety risks the demo-scale experiments of biohydrogen gas technology were moved from Biorefinery demo plant (Örnsköldsvik) of 40 m3 capacity to ATEX classified pilot-scale facility with 0.4 m3 capacity. Hence, bacterial biomass enough for the large-scale fish feed ingredient could not be produced. Lab-scale experiments with Caldicellulosiruptor cells as fish feed ingredient showed promising results as a protein-rich, sustainable fish feed ingredient. In addition, PHA biopolymer also showed favourable results as fish food ingredient for experiments at Gårdsfisk AB. Lab-scale experimental tests showed that the surplus activated sludge from the mills wastewater treatment could currently accumulate PHA to about 20 % of its dry weight. Mass balance evaluations based on realistically achievable expectations indicated a PHA biopolymer production potential of 3 600 tons of PHA per year from available organic residuals and for the two evaluated mills combined. 

The MultiBio concept has a positive climate impact in comparison with current treatment and moves developments in a positive direction to achieve 7 of the 10 Swedish environmental goals. Through a detailed feasibility analysis, a natural progression in next steps in scenarios were suggested for PHA production. The MultiBio cascade process can be implemented with further necessary development with good business potential and a positive effect on climate change. However, biohydrogen technology needs further developments before this cascade process concept can be implemented. Alternatively, a scenario with only biopolymer technology shows already a significant business potential and even larger positive effect on climate change. A successful next step in demonstration of the PHA biopolymer production scenario may lead to it being implemented within the next few years. Furthermore, MultiBio has attracted a lot of attention regionally and nationally but also internationally with a total of 65 media listings. A licentiate thesis and three university degree projects linked to the project have been completed. Overall, the MultiBio project has successfully achieved its goals and objectives.

MultiBio syftade till att etablera och demonstrera ett nytt bioraffinaderi-kaskadkoncept med tre förnybara biobaserade produkter: 1) bioväte, 2) biopolymerer och 3) proteinrika foderingredienser för fiskodling. Kaskadkonceptet utnyttjar förmågan hos en bakterie (Caldicellulosiruptor saccharolyticus) att omvandla näringsämnen som finns i massa- och pappersindustrins lågvärdiga processavloppsvatten till högvärdiga produkter vätgas, organiska syror och mikrobiell biomassa. Det utgående vattnet, rikt på organiska syror, hanteras sedan i en bioprocess med blandad mikrobiell kultur som används för att rena processvattnet och samtidigt producera biopolymerer av typen polyhydroxyalkanoater (PHA). Eftersom C. saccharolyticus proteininnehållet är mer än 63 % av celltorrvikt, utvärderades deras potential för beredning av fiskfoder.

En fiberslam-innehållande CTMP-restström visade sig vara en lämplig råvara för konceptet. På grund av säkerhetsrisker flyttades demoskalaexperimenten av biovätgasteknik från Biorefinery-demoanläggning (Örnsköldsvik) med 40 m3 kapacitet till ATEX-klassificerad pilotskaleanläggning med 0,4 m3 kapacitet. Därför kunde inte tillräckligt med bakteriebiomassa för den storskaliga fiskfoderingrediensen produceras. Experiment i laboratorieskala med Caldicellulosiruptor-celler som fiskfoderingrediens visade lovande resultat som en proteinrik, hållbar fiskfoderingrediens. Dessutom visade PHA-biopolymeren gynnsamma resultat som fiskfoderingrediens för experiment på Gårdsfisk AB. Experimentella test i laboratorieskala visade att bioslammet från bruken kunde ackumulera PHA till cirka 20 % av dess torrvikt. Massbalansbedömningar baserade på realistiska förväntningar indikerade en produktionspotential på 3 600 ton PHA per år från tillgängligt organiskt avfall vid de två ingående bruken. 

MultiBio-konceptet har en positiv klimatpåverkan jämfört med nuvarande behandling och har potential att bidra i rätt riktning för att uppnå 7 av de 10 svenska miljömålen. Genom en detaljerad genomförbarhetsanalys föreslogs scenarier med en stegvis implementering. MultiBio-kaskadprocessen kan implementeras med ytterligare nödvändig utveckling med god affärspotential och en positiv effekt på klimatförändringen. Men bioväte-tekniken behöver vidareutvecklas innan detta kaskad-koncept kan implementeras. Samtidigt visar ett scenario med enbart biopolymerteknologi redan nu en signifikant affärspotential och ännu större positiv effekt på klimatförändringen. En framgångsrik demonstration av det senare scenariot med endast PHA-produktion kan leda till att det genomförs inom de närmaste åren. Dessutom MultiBio har rönt stor uppmärksamhet regionalt och nationellt men även internationellt med totalt 65st medianoteringar. En licentiatavhandling och tre examensarbeten har färdigställts kopplat till projektet. Sammantaget har MultiBio framgångsrikt uppnått sina syften och mål.

Detta hypotestestförprojekt har haft som mål att validera potentialen för ett nyttmultifunktionellt kaskadbioraffinaderi som producerar fyra viktigaplattformsprodukter från ett Kraftverk: cellulosa, lignobränsle, biovätgas ochbiopolymer. Eftersom cellulosa- och ligninproduktionslinjer är väl karakteriseradefokuserar Bioplastogen på att utvärdera och identifiera möjligheter ochutmaningar vid vätgas- och bioplastproduktion från hemicellulosarestprodukter.Utförandet har varit indelat i tre faser dvs 1) identifierandet av hemicellulosarikaströmmar för biovätgasprocessen, 2) fermenteringexperiment för produktion avvätgas och organisk syra, 3) omvandling av organisk syra till PHA genomaktiverat slam. Valet av hemicellulosafraktion baserades på ett litteraturstudieoch genom diskussioner med personal på olika bruk och på LignoBoostpilotanläggning. Eftersom Lignoboost restprodukt har alternativaanvändningsområdet och hög halt inhibitorer togs ett beslut att användahemicellulosa som extraheras innan den blir en del av svartlut.Hemicellulosafraktionen bestod av 19g/L organiskt material varav sockerarternafrämst var pentoser. Fraktionens effekt på vätgasproduktionen utvärderas iserumflaskor och bioreaktorer med osmotoleranta stammar avCaldicellulosiruptor sacchrolyticus. Bioprocessen utvecklades även förindustriella applikation. Försök i serumflaskor visade potential förbiovätgasproduktion som inte kunde replikeras i reaktorsförsöken. Förslag på nystrategi för att förbättra biovätgasproduktionen också i reaktorförsken kan vara attantinger anpassa mikroorganismerna till de nya substraten och/eller att användablandkulturer som kan vara mer robusta mot denna typ av substrat.Den organiska biprodukten från biovätgasprocessen användes förbiopolymerproduktionsprocessen. Två olika slam från Sjölundavattenreningsanläggning användes och visade en bra potential genom attackumulera mer än 25% av gPHA/gVSS. Detta resultat stärkte projekthypotesen.Nästa steg blir att använda ett acklimatiserat slam från biologisk vattenrening frånett pappersmassabruk.Genom Bioplastogen har vi stärkt våra hypoteser men också identifieradestrategier för fortsatt förbättring som förhoppningsvis kan bli implementerade iansökt fortsättningsprojekt – HyPer

This hypothesis testing project has been with aim to validate potential for a novelmultipurpose cascading biorefinery, producing four principal platform productswithin a Kraft pulp mill: cellulose, lignofuels, biohydrogen and biopolymers.Cellulose and lignin product lines are presently well characterized at commercialscale. The project was, therefore, focused on evaluating and identifyingopportunities and challenges in processes for biohydrogen and biopolymersproduction from mill hemicellulose residuals.The project undertaking comprised three phases: determining the hemicelluloserich stream (HRS) to use, fermentation of sugars to hydrogen gas and organicacids, and conversion of organic acids to PHA by activated sludge. A literaturesurvey and discussions with the experts at various pulp and paper industries in theKarlstad region, as well as the developers of LignoBoost technology at Innventiaclearly mark the challenges of using black liquor directly as the HRS. Thesechallenges can be avoided by pre-extraction of the hemicellulose residual massvia ‘autohydrolysis’ of wood before the Kraft process. Hence, the residualhemicellulose before it enters the black liquor was the selected HRS.The HRS contained about 19 g/L of organics comprising of mainly pentose sugarsand organic acids along with some soluble lignin. A number of batch experimentswere performed in flasks that confirmed potential for H2 production by anosmotolerant strain (CSG5) of Caldicellulosiruptor sacchrolyticus. Experimentswere also performed in a fed-batch bioreactor. Here practical advancement wasmade with respect to process developments for industrial applications. However,no significant growth or H2 production was observed when permeate was added tothe reactor. This outcome was considered in hindsight to be due to an unforeseenoutcome of headspace partial pressure generated by the experimental set up.Nevertheless, given the HRS strategy and first positive indicators from batchexperiments, next steps to establish the industrial methods for conversion ofautohydrolysis hemicellulose residuals to H2 were recommended based onadaptation of the strain to the feed and/or application of co-culture bioprocessengineering.The effluent from biohydrogen production feeds a biopolymer production process.Two distinct sludge samples were sourced from municipal water treatment atSjölunda to evaluate polyhydroxyalkanoates (PHA) accumulation potential. Themixed cultures naturally accumulated a moderate level of more than 25% ofgPHA/gVSS. The conversion of the organic matter in the HRS stream into PHAwas successfully observed thus supporting the project hypothesis. Similarly to thebiohydrogen outcome, next steps will entail the development of an acclimatedenrichment PHA producing biomass from Kraft mill industry wastewaterbiological treatment.Through this work steps were made that strengthened the ideas as well as thestrategy forward which are to be implemented through the forthcoming proposedproject – HyPer.

Background: Caldicellulosiruptor species have gained a reputation as being among the best microorganisms to produce hydrogen (H2) due to possession of a combination of appropriate features. However, due to their low volumetric H2 productivities (Q H2), Caldicellulosiruptor species cannot be considered for any viable biohydrogen production process yet. In this study, we evaluate biofilm forming potential of pure and co-cultures of Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor owensensis in continuously stirred tank reactors (CSTR) and up-flow anaerobic (UA) reactors. We also evaluate biofilms as a means to retain biomass in the reactor and its influence on Q H2. Moreover, we explore the factors influencing the formation of biofilm. Results: Co-cultures of C. saccharolyticus and C. owensensis form substantially more biofilm than formed by C. owensensis alone. Biofilms improved substrate conversion in both of the reactor systems, but improved the Q H2 only in the UA reactor. When grown in the presence of each other's culture supernatant, both C. saccharolyticus and C. owensensis were positively influenced on their individual growth and H2 production. Unlike the CSTR, UA reactors allowed retention of C. saccharolyticus and C. owensensis when subjected to very high substrate loading rates. In the UA reactor, maximum Q H2 (approximately 20 mmol∈·∈L-1∈ ·∈h-1) was obtained only with granular sludge as the carrier material. In the CSTR, stirring negatively affected biofilm formation. Whereas, a clear correlation was observed between elevated (>40 μM) intracellular levels of the secondary messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) and biofilm formation. Conclusions: In co-cultures C. saccharolyticus fortified the trade of biofilm formation by C. owensensis, which was mediated by elevated levels of c-di-GMP in C. owensensis. These biofilms were effective in retaining biomass of both species in the reactor and improving Q H2 in a UA reactor using granular sludge as the carrier material. This concept forms a basis for further optimizing the Q H2 at laboratory scale and beyond. © 2015 Pawar et al.; licensee BioMed Central.

BACKGROUND: Caloramator celer is a strict anaerobic, alkalitolerant, thermophilic bacterium capable of converting glucose to hydrogen (H2), carbon dioxide, acetate, ethanol and formate by a mixed acid fermentation. Depending on the growth conditions C. celer can produce H2 at high yields. For a biotechnological exploitation of this bacterium for H2 production it is crucial to understand the factors that regulate carbon and electron fluxes and therefore the final distribution of metabolites to channel the metabolic flux towards the desired product.

RESULTS: Combining experimental results from batch fermentations with genome analysis, reconstruction of central carbon metabolism and metabolic flux analysis (MFA), this study shed light on glucose catabolism of the thermophilic alkalitolerant bacterium C. celer. Two innate factors pertaining to culture conditions have been identified to significantly affect the metabolic flux distribution: culture pH and partial pressures of H2 (PH2). Overall, at alkaline to neutral pH the rate of biomass synthesis was maximized, whereas at acidic pH the lower growth rate and the less efficient biomass formation are accompanied with more efficient energy recovery from the substrate indicating high cell maintenance possibly to sustain intracellular pH homeostasis. Higher H2 yields were associated with fermentation at acidic pH as a consequence of the lower synthesis of other reduced by-products such as formate and ethanol. In contrast, PH2 did not affect the growth of C. celer on glucose. At high PH2 the cellular redox state was balanced by rerouting the flow of carbon and electrons to ethanol and formate production allowing unaltered glycolytic flux and growth rate, but resulting in a decreased H2 synthesis.

CONCLUSION: C. celer possesses a flexible fermentative metabolism that allows redistribution of fluxes at key metabolic nodes to simultaneously control redox state and efficiently harvest energy from substrate even under unfavorable conditions (i.e. low pH and high PH2). With the H2 production in mind, acidic pH and low PH2 should be preferred for a high yield-oriented process, while a high productivity-oriented process can be achieved at alkaline pH and high PH2.