Characterization and adaptation of Caldicellulosiruptor strains to higher sugar concentrations, targeting enhanced hydrogen production from lignocellulosic hydrolysates

Background The members of the genus Caldicellulosiruptor have the potential for future integration into a biorefinery system due to their capacity to generate hydrogen close to the theoretical limit of 4 mol H2/mol hexose, use a wide range of sugars and can grow on numerous lignocellulose hydrolysates. However, members of this genus are unable to survive in high sugar concentrations, limiting their ability to grow on more concentrated hydrolysates, thus impeding their industrial applicability. In this study five members of this genus, C.owensensis, C. kronotskyensis, C.bescii, C.acetigenus and C.kristjanssonii, were developed to tolerate higher sugar concentrations through an adaptive laboratory evolution (ALE) process. The developed mixed population C.owensensis CO80 was further studied and accompanied by the development of a kinetic model based on Monod kinetics to quantitatively compare it with the parental strain. Results Mixed populations of Caldicellulosiruptor tolerant to higher glucose concentrations were obtained with C.owensensis adapted to grow up to 80 g/L glucose; other strains in particular C. kristjanssonii demonstrated a greater restriction to adaptation. The C.owensensis CO80 mixed population was further studied and demonstrated the ability to grow in glucose concentrations up to 80 g/L glucose, but with reduced volumetric hydrogen productivities ($$Q_{{{\text{H}}_{2} }}$$ Q H 2 ) and incomplete sugar conversion at elevated glucose concentrations. In addition, the carbon yield decreased with elevated concentrations of glucose. The ability of the mixed population C.owensensis CO80 to grow in high glucose concentrations was further described with a kinetic growth model, which revealed that the critical sugar concentration of the cells increased fourfold when cultivated at higher concentrations. When co-cultured with the adapted C.saccharolyticus G5 mixed culture at a hydraulic retention time (HRT) of 20 h, C.owensensis constituted only 0.09–1.58% of the population in suspension. Conclusions The adaptation of members of the Caldicellulosiruptor genus to higher sugar concentrations established that the ability to develop improved strains via ALE is species dependent, with C.owensensis adapted to grow on 80 g/L, whereas C.kristjanssonii could only be adapted to 30 g/L glucose. Although C.owensensis CO80 was adapted to a higher sugar concentration, this mixed population demonstrated reduced $$Q_{{{\text{H}}_{2} }}$$ Q H 2 with elevated glucose concentrations. This would indicate that while ALE permits adaptation to elevated sugar concentrations, this approach does not result in improved fermentation performances at these higher sugar concentrations. Moreover, the observation that planktonic mixed culture of CO80 was outcompeted by an adapted C.saccharolyticus, when co-cultivated in continuous mode, indicates that the robustness of CO80 mixed culture should be improved for industrial application.

Identification of the major fermentation inhibitors of recombinant 2G yeasts in diverse lignocellulose hydrolysates

Background Presence of inhibitory chemicals in lignocellulose hydrolysates is a major hurdle for production of second-generation bioethanol. Especially cheaper pre-treatment methods that ensure an economical viable production process generate high levels of these inhibitory chemicals. The effect of several of these inhibitors has been extensively studied with non-xylose-fermenting laboratory strains, in synthetic media, and usually as single inhibitors, or with inhibitor concentrations much higher than those found in lignocellulose hydrolysates. However, the relevance of individual inhibitors in inhibitor-rich lignocellulose hydrolysates has remained unclear. Results The relative importance for inhibition of ethanol fermentation by two industrial second-generation yeast strains in five lignocellulose hydrolysates, from bagasse, corn cobs and spruce, has now been investigated by spiking higher concentrations of each compound in a concentration range relevant for industrial hydrolysates. The strongest inhibition was observed with industrially relevant concentrations of furfural causing partial inhibition of both D-glucose and D-xylose consumption. Addition of 3 or 6 g/L furfural strongly reduced the ethanol titer obtained with strain MD4 in all hydrolysates evaluated, in a range of 34 to 51% and of 77 to 86%, respectively. This was followed by 5-hydroxymethylfurfural, acetic acid and formic acid, for which in general, industrially relevant concentrations caused partial inhibition of D-xylose fermentation. On the other hand, spiking with levulinic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid or vanillin caused little inhibition compared to unspiked hydrolysate. The further evolved MD4 strain generally showed superior performance compared to the previously developed strain GSE16-T18. Conclusion The results highlight the importance of individual inhibitor evaluation in a medium containing a genuine mix of inhibitors as well as the ethanol that is produced by the fermentation. They also highlight the potential of increasing yeast inhibitor tolerance for improving industrial process economics.

Characterization and Development of Osmotolerant Caldicellulosiruptor Strains Targeting Enhanced Hydrogen Production from Lignocellulosic Hydrolysates

Background The members of the genus Caldicellulosiruptor have the potential for future integration into a biorefinery system due to their capacity to generate hydrogen close to the theoretical limit of 4 mol H 2 /mol hexose, use a wide range of sugars and can grow on numerous lignocellulose hydrolysates. However, members of this genus are unable to survive in high osmolarity conditions, limiting their ability to grow on more concentrated hydrolysates, thus impeding their industrial applicability. In this study five members of this genus, C. owensensis , C. kronotskyensis , C. bescii, C. acetigenus and C. kristjanssonii , were developed to tolerate higher osmolarities through an adaptive laboratory evolution (ALE) process. The developed strain C. owensensis CO80 was further studied accompanied by the development of a kinetic model based on Monod kinetics. Results Osmotolerant strains of Caldicellulosiruptor were obtained with C. owensensis adapted to grow up to 80 g/l glucose; other strains in particular C. kristjanssonii demonstrated a greater restriction to adaptation. C. owensensis CO80 was further studied and demonstrated the ability to grow in glucose concentrations up to 80 g/l glucose but with reduced volumetric hydrogen productivities (Q H2 ) and incomplete sugar conversion at elevated glucose concentrations. In addition, the carbon yield decreased with elevated concentrations of glucose. The ability of C. owensensis CO80 to grow in high glucose concentrations was further described with a kinetic growth model, which revealed that the critical osmolarity of the cells increased fourfold when cultivated at higher osmolarity. When co-cultured with the osmotolerant strain C. saccharolyticus G5 at a hydraulic retention time (HRT) of 20h, C. owensensis constituted only 0.09-1.58% of the population in suspension.Conclusions The adaptation of members of the Caldicellulosiruptor genus to higher osmolarity established that the ability to develop improved strains via ALE is species dependent, with C. owensensis adapted to grow on 80 g/l, whereas C. kristjanssonii could only be adapted to 30 g/l glucose. Although, C. owensensis CO80 was adapted to a higher osmolarity medium, the strain demonstrated reduced Q H2 with elevated glucose concentrations. This would indicate that while ALE permits adaptation to elevated osmolarities, this approach does not result in improved fermentation performances at these higher osmolarities. Moreover, the observation that planktonic culture of CO80 was outcompeted by an osmotolerant strain of C. saccharolyticus, when co-cultivated in continuous mode, indicates that the robustness of strain CO80 should be improved for industrial application .

Engineered Thermoanaerobacterium aotearoense with nfnAB knockout for improved hydrogen production from lignocellulose hydrolysates

Background As a renewable and clean energy carrier, the production of biohydrogen from low-value feedstock such as lignocellulose has increasingly garnered interest. The NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB) complex catalyzes electron transfer between reduced ferredoxin and NAD(P)+, which is critical for production of NAD(P)H-dependent products such as hydrogen and ethanol. In this study, the effects on end-product formation of deletion of nfnAB from Thermoanaerobacterium aotearoense SCUT27 were investigated. Results Compared with the parental strain, the NADH/NAD+ ratio in the ∆nfnAB mutant was increased. The concentration of hydrogen and ethanol produced increased by (41.1 ± 2.37)% (p < 0.01) and (13.24 ± 1.12)% (p < 0.01), respectively, while the lactic acid concentration decreased by (11.88 ± 0.96)% (p < 0.01) when the ∆nfnAB mutant used glucose as sole carbon source. No obvious inhibition effect was observed for either SCUT27 or SCUT27/∆nfnAB when six types of lignocellulose hydrolysate pretreated with dilute acid were used for hydrogen production. Notably, the SCUT27/∆nfnAB mutant produced 190.63–209.31 mmol/L hydrogen, with a yield of 1.66–1.77 mol/mol and productivity of 12.71–13.95 mmol/L h from nonsterilized rice straw and corn cob hydrolysates pretreated with dilute acid. Conclusions The T. aotearoense SCUT27/∆nfnAB mutant showed higher hydrogen yield and productivity compared with those of the parental strain. Hence, we demonstrate that deletion of nfnAB from T. aotearoense SCUT27 is an effective approach to improve hydrogen production by redirecting the electron flux, and SCUT27/∆nfnAB is a promising candidate strain for efficient biohydrogen production from lignocellulosic hydrolysates.