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.

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.

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.

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.

BACKGROUND: Caldicellulosiruptor saccharolyticus 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 (PH2) is a critical aspect to eliminate the need for continuous stripping of the produced H2 from the bioreactor.

RESULTS: Herein, we demonstrate that, under given conditions, growth and H2 production in C. saccharolyticus can be sustained at PH2 up to 67 kPa in a chemostat. At this PH2, 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 (Ki 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 C. saccharolyticus 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 C. saccharolyticus to grow and produce H2 under relatively high PH2.

CONCLUSION: C. saccharolyticus is able to grow and produce hydrogen at high PH2, 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 H2 yield.

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.