Key role of K+ and Ca2+ in high-yield ethanol production by S. Cerevisiae from concentrated sugarcane molasses.

BACKGROUND: Saccharomyces cerevisiae is an important microorganism in ethanol synthesis, and with sugarcane molasses as the feedstock, ethanol is being synthesized sustainably to meet growing demands. However, high-concentration ethanol fermentation based on high-concentration sugarcane molasses-which is needed for reduced energy consumption of ethanol distillation at industrial scale-is yet to be achieved. RESULTS: In the present study, to identify the main limiting factors of this process, adaptive laboratory evolution and high-throughput screening (Py-Fe3+) based on ARTP (atmospheric and room-temperature plasma) mutagenesis were applied. We identified high osmotic pressure, high temperature, high alcohol levels, and high concentrations of K+, Ca2+, K+ and Ca2+ (K+&Ca2+), and sugarcane molasses as the main limiting factors. The robust S. cerevisiae strains of NGT-F1, NGW-F1, NGC-F1, NGK+, NGCa2+ NGK+&Ca2+-F1, and NGTM-F1 exhibited high tolerance to the respective limiting factor and exhibited increased yield. Subsequently, ethanol synthesis, cell morphology, comparative genomics, and gene ontology (GO) enrichment analysis were performed in a molasses broth containing 250 g/L total fermentable sugars (TFS). Additionally, S. cerevisiae NGTM-F1 was used with 250 g/L (TFS) sugarcane molasses to synthesize ethanol in a 5-L fermenter, giving a yield of 111.65 g/L, the conversion of sugar to alcohol reached 95.53%. It is the highest level of physical mutagenesis yield at present. CONCLUSION: Our results showed that K+ and Ca2+ ions primarily limited the efficient production of ethanol. Then, subsequent comparative transcriptomic GO and pathway analyses showed that the co-presence of K+ and Ca2+ exerted the most prominent limitation on efficient ethanol production. The results of this study might prove useful by promoting the development and utilization of green fuel bio-manufactured from molasses.

A Self-Assembling γPFD-SpyCatcher Hydrogel Scaffold for the Coimmobilization of SpyTag-Enzymes to Facilitate the Catalysis of Regulated Enzymes.

In this study, a γPFD-SpyCatcher hydrogel scaffold with the capacity for spontaneous assembly was established. With a maximum loading capacity of a 1:1 molar ratio with SpyTag-enzymes, the immobilized proteins can not only rapidly provide pure enzymes but also exhibit improved thermal and pH stability. The results of the transmission electron microscopic analysis and the traits they present indicated that SpyCatcher promotes the aggregation of γPFD and the formation of hydrogels. In the cell-free pyruvate synthesis system, the γPFD-SpyCatcher coimmobilized SpyTag-hexokinase (HK), SpyTag-phosphofructokinase (PFK) and SpyTag-pyruvate kinase (PK) were employed, and the production of pyruvate increased by 43, 78 and 47% respectively. In in vitro experiments, the oxidative deamination activity of glutamate dehydrogenase (GDH) coimmobilized with γPFD-SpyCatcher was 38% higher than that of purified enzymes. These findings indicate that the γPFD-SpyCatcher-based hydrogels play an important role in breaking the barrier of regulatory enzymes and will provide more strategies for the development of synthetic biology.

Nitrogen-doped porous carbon encapsulates multivalent cobalt-nickel as oxygen reduction reaction catalyst for zinc-air battery.

In this study, we present a bimetallic ion coexistence encapsulation strategy employing hexadecyl trimethyl ammonium bromide (CTAB) as a mediator to anchor cobalt-nickel (CoNi) bimetals in nitrogen-doped porous carbon cubic nanoboxes (CoNi@NC). The fully encapsulated and uniformly dispersed CoNi nanoparticles with the improved density of active sites help to accelerate the oxygen reduction reaction (ORR) kinetics and provide an efficient charge/mass transport environment. Zinc-air battery (ZAB) equipped CoNi@NC as cathode exhibits an open-circuit voltage of 1.45 V, a specific capacity of 870.0 mAh g-1, and a power density of 168.8 mW cm-2. Moreover, the two CoNi@NC-based ZABs in series display a stable discharge specific capacity of 783.0 mAh g-1, as well as a large peak power density of 387.9 mW cm-2. This work provides an effective way to tune the dispersion of nanoparticles to boost active sites in nitrogen-doped carbon structure, and enhance the ORR activity of bimetallic catalysts.