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.

Genome-wide Association Study of Individual Sugar Content and Sugar Conversion in Fruit of Japanese Pear (Pyrus spp.)

Background: Sweetness is one of the most important traits determining fruit quality. Sweetness is controlled not only by the total sugar content but also by the contents of individual sugars. The major sugars in mature Rosaceae fruits are sucrose, fructose, glucose, and sorbitol, which have different levels of sweetness. Among these, sucrose and fructose have high sweetness, whereas glucose and sorbitol have low sweetness. The objective of this study was to identify the quantitative trait loci (QTLs) associated with fruit traits including individual sugar accumulation and conversion, to infer the candidate genes underlying the QTLs, and to assess the potential of genomic selection for breeding pear fruit traits.Results: We evaluated 10 fruit traits and conducted genome-wide association studies (GWAS) for 106 cultivars and 17 breeding populations (1112 F1 individuals) using 3484 tag single-nucleotide polymorphisms (SNPs) genotyped by double-digest restriction-site associated DNA sequencing (ddRAD-Seq). By implementing a mixed linear model and a Bayesian multiple-QTL model in GWAS, 56 SNPs associated with fruit traits were identified. Four loci were presumed to be associated with sugar conversion because the SNPs were significant for more than one individual sugar and the individual sugar contents associated with each SNP genotype were negatively correlated. In particular, a SNP located close to acid invertase gene PPAIV3 on chromosome 7 and a newly identified SNP on chromosome 11 had quite large effects on sugar conversion. We used ‘Golden Delicious’ doubled haploid (GDDH) 13, an apple reference genome to infer the candidate genes for the identified SNPs. In the region flanking the SNP on chromosome 11, there is a tandem repeat of early responsive to dehydration (ERD6)-like sugar transporter genes which might play a role in the phenotypes observed.Conclusions: SNPs associated with sugar accumulation and conversion were newly identified at several loci, and candidate genes underlying QTLs were inferred using advanced apple genome information. Several QTLs showed clear effects with more than 10% of phenotypic variance explained by those SNPs in the breeding populations. By combining the effects of multiple QTLs, breeders would be able to select seedlings that will later bear fruit with high sucrose and fructose content.

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 .