Short-term adaptation as a tool to improve bioethanol production using grass press-juice as fermentation medium.

Grass raw materials collected from grasslands cover more than 30% of Europe's agricultural area. They are considered very attractive for the production of different biochemicals and biofuels due to their high availability and renewability. In this study, a perennial ryegrass (Lolium perenne) was exploited for second-generation bioethanol production. Grass press-cake and grass press-juice were separated using mechanical pretreatment, and the obtained juice was used as a fermentation medium. In this work, Saccharomyces cerevisiae was utilized for bioethanol production using the grass press-juice as the sole fermentation medium. The yeast was able to release about 11 g/L of ethanol in 72 h, with a total production yield of 0.38 ± 0.2 gEthanol/gsugars. It was assessed to improve the fermentation ability of Saccharomyces cerevisiae by using the short-term adaptation. For this purpose, the yeast was initially propagated in increasing the concentration of press-juice. Then, the yeast cells were re-cultivated in 100%(v/v) fresh juice to verify if it had improved the fermentation efficiency. The fructose conversion increased from 79 to 90%, and the ethanol titers reached 18 g/L resulting in a final yield of 0.50 ± 0.06 gEthanol/gsugars with a volumetric productivity of 0.44 ± 0.00 g/Lh. The overall results proved that short-term adaptation was successfully used to improve bioethanol production with S. cerevisiae using grass press-juice as fermentation medium. KEY POINTS: • Mechanical pretreatment of grass raw materials • Production of bioethanol using grass press-juice as fermentation medium • Short-term adaptation as a tool to improve the bioethanol production.

Co-cultivation of diazotrophic terrestrial cyanobacteria and Arabidopsis thaliana.

Diazotrophic cyanobacteria are able to fix N2 from the atmosphere and release it as bioavailable nitrogen what other organisms can utilize. Thus, they could be used as living nitrogen supplier whereby the use of fertilizer could be reduced in agricultural industry what results in a decrease of laughing gas released during fertilizer production. The diazotroph cyanobacterium Desmonostoc muscorum (D. muscorum) was characterized in shake flasks cultivated in nitrogen-free and nitrogen-containing medium. Similar growth rates were reached in both cultivations and the release of ammonium by D. muscorum was detected under nitrogen depletion. Subsequently, D. muscorum was co-cultivated with Arabidopsis thaliana (A. thaliana) in nitrogen-free medium. Additionally, the plant was cultivated in nitrogen containing and nitrogen-free medium without D. muscorum as reference. A co-cultivation led to higher growth rates of the cyanobacterium and similar growth of A. thaliana with similar maximum photochemical efficiency of photosystem II compared to the growth of nitrogen containing medium. Further, accumulation of cyanobacterial cells around the roots of A. thaliana was detected, indicating a successfully induced artificial symbiosis. Based on these results, D. muscorum could be a promising cyanobacterium as living nitrogen supplier for plants.

Pink bacteria-Production of the pink chromophore phycoerythrobilin with Escherichia coli.

Phycoerythrobilin (PEB) is an open-chain tetrapyrrole derived from heme and plays an important role as light-harvesting pigment in the phycobiliproteins of cyanobacteria and red algae. Furthermore, PEB can also function as an antioxidant with potential use as a natural acid stable food colorant. PEB is not commercially available and large, pure quantities can only be obtained by laborious methanolysis of red algae followed by liquid chromatography. Here we describe an improved method for high yield production and purification of PEB in Escherichia coli via heterologous expression where the two required enzymes heme oxygenase and PEB synthase subsequently convert the substrate heme provided by the host cell. Experiments in shaking flasks resulted in the highest product yield of 680.23 ±â€¯42.75 µg PEB per g cell dry weight, by induction with 0.1 mM IPTG. Scale-up to batch-operated fermentation in a 2 L bioreactor reached product concentrations up to 5.02 mg PEB L-1 by adjustment of aeration, induction time, media composition and supplementation of precursors. A further approach included separation of PEB from developed foam above the culture. This enabled continuous product collection during cultivation and simplified product purification. Produced PEB was validated via UV-vis spectroscopy, high pressure liquid chromatography and mass spectrometry.

Determination of triterpenic acids and screening for valuable secondary metabolites in Salvia sp. suspension cultures.

Plant in vitro cultures are a prospective alternative for biochemicals production, for example the triterpenes oleanolic and ursolic acid present in plants and cell cultures of Salvia sp. Our objective was to develop a suitable analysis protocol for evaluation of triterpenic acid yield in plant raw material and in vitro cultures supporting selection processes. Moreover, valuable bioactive compounds had to be revealed. Thus, different strategies enhancing the separation for a sensitive and effective HPLC-UV method were investigated and the developed method was validated for linearity, precision, accuracy, limits of detection and quantification. A baseline separation of these isomers enabled detection limits of below 0.4 microg/mL and quantification limits of about 1.2 microg/mL. Over the tested concentration range a good linearity was observed (R2 > 0.9999). The variations in the method were below 6% for intra- and inter-day assays of concentration. Recoveries were between 85-98% for both compounds using ethanol as extraction solvent. Additionally, metabolite profiling of cell suspension culture extracts by GC-MS has shown the production variability of different plant metabolites and especially the presence of plant phenols and sterols. These studies provide a method suitable for screening plant and cell culture productivity of triterpenic acids and highlighted interesting co-products of plant cell cultures.