Reassessment of hydrogen tolerance in Caldicellulosiruptor saccharolyticus

<p>Abstract</p> <p>Background</p> <p><it>Caldicellulosiruptor saccharolyticus </it>has the ability to produce hydrogen (H₂) at high yields from a wide spectrum of carbon sources, and has therefore gained industrial interest. For a cost-effective biohydrogen process, the ability of an organism to tolerate high partial pressures of H₂(<it>P</it>_H2) is a critical aspect to eliminate the need for continuous stripping of the produced H₂from the bioreactor.</p> <p>Results</p> <p>Herein, we demonstrate that, under given conditions, growth and H₂production in <it>C. saccharolyticus </it>can be sustained at <it>P</it>_H2up to 67 kPa in a chemostat. At this <it>P</it>_H2, 38% and 16% of the pyruvate flux was redirected to lactate and ethanol, respectively, to maintain a relatively low cytosolic NADH/NAD ratio (0.12 mol/mol). To investigate the effect of the redox ratio on the glycolytic flux, a kinetic model describing the activity of the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was developed. Indeed, at NADH/NAD ratios of 0.12 mol/mol (<it>K</it>i of NADH = 0.03 ± 0.01 mM) GAPDH activity was inhibited by only 50% allowing still a high glycolytic flux (3.2 ± 0.4 mM/h). Even at high NADH/NAD ratios up to 1 mol/mol the enzyme was not completely inhibited. During batch cultivations, hydrogen tolerance of <it>C. saccharolyticus </it>was dependent on the growth phase of the organism as well as the carbon and energy source used. The obtained results were analyzed, based on thermodynamic and enzyme kinetic considerations, to gain insight in the mechanism underlying the unique ability of <it>C. saccharolyticus </it>to grow and produce H₂under relatively high <it>P</it>_H2.</p> <p>Conclusion</p> <p><it>C. saccharolyticus </it>is able to grow and produce hydrogen at high <it>P</it>_H2, hence eliminating the need of gas sparging in its cultures. Under this condition, it has a unique ability to fine tune its metabolism by maintaining the glycolytic flux through regulating GAPDH activity and redistribution of pyruvate flux. Concerning the later, xylose-rich feedstock should be preferred over the sucrose-rich one for better H₂yield.</p

Biofilm formation by designed co-cultures of Caldicellulosiruptor species as a means to improve hydrogen productivity

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 (&gt;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

Effect of nitrogen availability on the poly-3-d-hydroxybutyrate accumulation by engineered Saccharomyces cerevisiae

Poly-3-d-hydroxybutyrate (or PHB) is a polyester which can be used in the production of biodegradable plastics from renewable resources. It is naturally produced by several bacteria as a response to nutrient starvation in the excess of a carbon source. The yeast Saccharomyces cerevisiae could be an alternative production host as it offers good inhibitor tolerance towards weak acids and phenolic compounds and does not depolymerize the produced PHB. As nitrogen limitation is known to boost the accumulation of PHB in bacteria, the present study aimed at investigating the effect of nitrogen availability on PHB accumulation in two recombinant S. cerevisiae strains harboring different xylose consuming and PHB producing pathways: TMB4443 expressing an NADPH-dependent acetoacetyl-CoA reductase and a wild-type S. stipitis XR with preferential use of NADPH and TMB4425 which expresses an NADH-dependent acetoacetyl-CoA reductase and a mutated XR with a balanced affinity for NADPH/NADH. TMB4443 accumulated most PHB under aerobic conditions and with glucose as sole carbon source, whereas the highest PHB concentrations were obtained with TMB4425 under anaerobic conditions and xylose as carbon source. In both cases, the highest PHB contents were obtained with high availability of nitrogen. The major impact of nitrogen availability was observed in TMB4425, where a 2.7-fold increase in PHB content was obtained. In contrast to what was observed in natural PHB-producing bacteria, nitrogen deficiency did not improve PHB accumulation in S. cerevisiae. Instead the excess available carbon from xylose was shunted into glycogen, indicating a significant gluconeogenic activity on xylose

Bio‑hydrogen and VFA production from steel mill gases using pure and mixed bacterial cultures

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: Environmental services from a multipurpose biorefinery

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.This report summarizes key developments made within the project MultiBio (Dnr 2017-03286) that was financed by Vinnova. MultiBio (Dnr 2017-03286

Abstracts of National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020

This book presents the abstracts of the papers presented to the Online National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020 (RDMPMC-2020) held on 26th and 27th August 2020 organized by the Department of Metallurgical and Materials Science in Association with the Department of Production and Industrial Engineering, National Institute of Technology Jamshedpur, Jharkhand, India. Conference Title: National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020Conference Acronym: RDMPMC-2020Conference Date: 26–27 August 2020Conference Location: Online (Virtual Mode)Conference Organizer: Department of Metallurgical and Materials Engineering, National Institute of Technology JamshedpurCo-organizer: Department of Production and Industrial Engineering, National Institute of Technology Jamshedpur, Jharkhand, IndiaConference Sponsor: TEQIP-