Use of Raman and Raman optical activity to extract atomistic details of saccharides in aqueous solution

Sugars are crucial components in biosystems and industrial applications. In aqueous environments, the natural state of short saccharides or charged glycosaminoglycans is floating and wiggling in solution. Therefore, tools to characterize their structure in a native aqueous environment are crucial but not always available. Here, we show that a combination of Raman/ROA and, on occasions, NMR experiments with Molecular Dynamics (MD) and Quantum Mechanics (QM) is a viable method to gain insights into structural features of sugars in solutions. Combining these methods provides information about accessible ring puckering conformers and their proportions. It also provides information about the conformation of the linkage between the sugar monomers, i.e., glycosidic bonds, allowing for identifying significantly accessible conformers and their relative abundance. For mixtures of sugar moieties, this method enables the deconvolution of the Raman/ROA spectra to find the actual amounts of its molecular constituents, serving as an effective analytical technique. For example, it allows calculating anomeric ratios for reducing sugars and analyzing more complex sugar mixtures to elucidate their real content. Altogether, we show that combining Raman/ROA spectroscopies with simulations is a versatile method applicable to saccharides. It allows for accessing many features with precision comparable to other methods routinely used for this task, making it a viable alternative. Furthermore, we prove that the proposed technique can scale up by studying the complicated Raffinose trisaccharide, and therefore, we expect its wide adoption to characterize sugar structural features in solution.

Potentials of utilizing renewable resources of agriculture residues for the biosynthesis of bacterial cellulose nanofibers

In the present study, production of bacterial cellulose nanofibers from single sugars and sugar mixtures in feedstock was investigated using different culture methods. This includes production in shake flask, stirred tank and draft tube airlift bioreactors. There has been no previous work done other than this reported work concerning production of bacterial cellulose nanofibers using sugar mixtures. Fructose was found to be the top producer with 5.65 g/L of bacterial cellulose nanofibers. The different compositions of sugar mixtures tested in this study were identical to acid hydrolyzates of agricultural residues. Maximum yield of 17.72 g/L was observed with mixture that resembles the acid hydrolyzate of wheat straw. Modified stirred tank and airlift bioreactors showed higher production of bacterial cellulose nanofibers compared to shake flask. In general, results obtained in the present study demonstrate potential of improving production of nanofibers solely based on agricultural residues and using draft tube airlift bioreactors.

Potentials of utilizing renewable resources of agriculture residues for the biosynthesis of bacterial cellulose nanofibers

In the present study, production of bacterial cellulose nanofibers from single sugars and sugar mixtures in feedstock was investigated using different culture methods. This includes production in shake flask, stirred tank and draft tube airlift bioreactors. There has been no previous work done other than this reported work concerning production of bacterial cellulose nanofibers using sugar mixtures. Fructose was found to be the top producer with 5.65 g/L of bacterial cellulose nanofibers. The different compositions of sugar mixtures tested in this study were identical to acid hydrolyzates of agricultural residues. Maximum yield of 17.72 g/L was observed with mixture that resembles the acid hydrolyzate of wheat straw. Modified stirred tank and airlift bioreactors showed higher production of bacterial cellulose nanofibers compared to shake flask. In general, results obtained in the present study demonstrate potential of improving production of nanofibers solely based on agricultural residues and using draft tube airlift bioreactors.

In Vitro Prebiotic and Anti-Colon Cancer Activities of Agar-Derived Sugars from Red Seaweeds

Numerous health benefits of diets containing red seaweeds or agar-derived sugar mixtures produced by enzymatic or acid hydrolysis of agar have been reported. However, among various agar-derived sugars, the key components that confer health-beneficial effects, such as prebiotic and anti-colon cancer activities, remain unclear. Here, we prepared various agar-derived sugars by multiple enzymatic reactions using an endo-type and an exo-type of β-agarase and a neoagarobiose hydrolase and tested their in vitro prebiotic and anti-colon cancer activities. Among various agar-derived sugars, agarotriose exhibited prebiotic activity that was verified based on the fermentability of agarotriose by probiotic bifidobacteria. Furthermore, we demonstrated the anti-colon cancer activity of 3,6-anhydro-l-galactose, which significantly inhibited the proliferation of human colon cancer cells and induced their apoptosis. Our results provide crucial information regarding the key compounds derived from red seaweeds that confer beneficial health effects, including prebiotic and anti-colon cancer activities, to the host.

Characterization of simultaneous uptake of xylose and glucose in Caldicellulosiruptor kronotskyensis for optimal hydrogen production

Background Caldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profiles of C. kronotskyensis in the presence of mixtures of glucose–xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study, we aimed to investigate further whether C. kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose). Results Interestingly, growth of C. kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus, C. kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C. kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μmax = 0.57 h−1) as on glucose (μmax = 0.28 h−1). A study of the sugar uptake was made with different glucose–xylose ratios to find a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be KI,glu = 0.01 cmol L−1 and KI,xyl = 0.001 cmol L−1, hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatics analysis could not exclude that C. kronotskyensis possesses more than one transporter for glucose. As a next step it was investigated whether glucose uptake by C. kronotskyensis improved in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose; nevertheless, the growth rate on each sugar remained similar. Conclusions C. kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C. kronotskyensis has adapted more strongly to pentose rather than hexose, thereby having obtained a specific survival edge in thermophilic lignocellulosic degradation communities.

Butanol production from lignocellulosic sugars by Clostridium beijerinckii in microbioreactors

Background Butanol (n-butanol) has been gaining attention as a renewable energy carrier and an alternative biofuel with superior properties to the most widely used ethanol. We performed 48 anaerobic fermentations simultaneously with glucose and xylose as representative lignocellulosic sugars by Clostridium beijerinckii NCIMB 8052 in BioLector® microbioreactors to understand the effect of different sugar mixtures on fermentation and to demonstrate the applicability of the micro-cultivation system for high-throughput anaerobic cultivation studies. We then compared the results to those of similar cultures in serum flasks to provide insight into different setups and measurement methods. Results ANOVA results showed that the glucose-to-xylose ratio affects both growth and production due to Carbon Catabolite Repression. The study demonstrated successful use of BioLector® system for the first time for screening several media and sugar compositions under anaerobic conditions by using online monitoring of cell mass and pH in real-time and at unprecedented time-resolution. Fermentation products possibly interfered with dissolved oxygen (DO) measurements, which require a careful interpretation of DO monitoring results. Conclusions The statistical approach to evaluate the microbioreactor setup, and information obtained in this study will support further research in bioreactor and bioprocess design, which are very important aspects of industrial fermentations of lignocellulosic biomass.

Characterization of Simultaneous Uptake of Xylose and Glucose in Caldicellulosiruptor Kronotskyensis for Optimal Hydrogen Production

BackgroundCaldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profiles of C . kronotskyensis in the presence of mixtures of glucose-xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study we aimed to investigated further whether C . kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose). ResultsInterestingly, growth of C . kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus , C . kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C . kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μ max = 0.57 h -1 ) as on glucose (μ max = 0.28 h -1 ). It was found that C . kronotskyensis takes up glucose and xylose simultaneously with the same transporter. A study of the sugar uptake was made with different glucose-xylose ratios to find a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be K I,glu = 0.01 cmol·L -1 and K I,xyl = 0.001 cmol·L -1 , hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatic analysis indicated the lack of another sugar uptake system in C . kronotskyensis as compared to C. saccharolyticus . Therefore, it was investigated whether glucose uptake by C . kronotskyensis was in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose, nevertheless, the growth rate on each sugar remained similar. ConclusionsC . kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C . kronotskyensis has adapted more strongly to pentose rather than hexose thereby having obtained a specific survival edge in thermophilic lignocellulosic degradation communities.

Dynamic Co-Cultivation Process of Corynebacterium glutamicum Strains for the Fermentative Production of Riboflavin

Residual streams from lignocellulosic processes contain sugar mixtures of glucose, xylose, and mannose. Here, the industrial workhorse Corynebacterium glutamicum was explored as a research platform for the rational utilization of a multiple sugar substrate. The endogenous manA gene was overexpressed to enhance mannose utilization. The overexpression of the xylA gene from Xanthomonas campestris in combination with the endogenous xylB gene enabled xylose consumption by C. glutamicum. Furthermore, riboflavin production was triggered by overexpressing the sigH gene from C. glutamicum. The resulting strains were studied during batch fermentations in flasks and 2 L lab-scale bioreactors separately using glucose, mannose, xylose, and a mixture of these three sugars as a carbon source. The production of riboflavin and consumption of sugars were improved during fed-batch fermentation thanks to a dynamic inoculation strategy of manA overexpressing strain and xylAB overexpressing strain. The final riboflavin titer, yield, and volumetric productivity from the sugar mixture were 27 mg L−1, 0.52 mg g−1, and 0.25 mg L−1 h−1, respectively. It reached a 56% higher volumetric productivity with 45% less by-product formation compared with an equivalent process inoculated with a single strain overexpressing the genes xylAB and manA combined. The results indicate the advantages of dynamic multi strains processes for the conversion of sugar mixtures.