Sequential Production of ᴅ-xylonate and Ethanol from Non-Detoxified Corncob at Low-pH by Pichia kudriavzevii via a Two-Stage Fermentation Strategy

Improving the comprehensive utilization of sugars in lignocellulosic biomass is a major challenge for enhancing the economic viability of lignocellulose biorefinement. A robust yeast Pichia kudriavzevii N-X showed excellent performance in ethanol production under high temperature and low pH conditions and was engineered for ᴅ-xylonate production without xylitol generation. The recombinant strain P. kudriavzevii N-X/S1 was employed for sequential production of ᴅ-xylonate and ethanol from ᴅ-xylose, feeding on ᴅ-glucose without pH control in a two-stage strategy of aerobic and shifting micro-aerobic fermentation. Acid-pretreated corncob without detoxification and filtration was used for ᴅ-xylonate production, then simultaneous saccharification and ethanol fermentation was performed with cellulase added at pH 4.0 and at 40 °C. By this strategy, 33.5 g/L ᴅ-xylonate and 20.8 g/L ethanol were produced at yields of 1.10 g/g ᴅ-xylose and 84.3% of theoretical value, respectively. We propose a promising approach for the sequential production of ᴅ-xylonate and ethanol from non-detoxified corncob using a single microorganism.

The Impact of the Fermentation Strategy on the Flavour Formation of Ilzer Rose (Malus domestica Borkh.) Apple Wine

The flavour and the volatilome of apple wines made from the Austrian heritage variety Ilzer Rose was in the scope of this study. The apple wines were produced by adopting oenological practises that are not commonly used in fruit wine production. Different fermentation strategies including the addition of enzymes with β-glucosidase activity, addition of a fining agent, maceration of the mash along with mash fermentation were applied. The volatile compounds of the juices as intermediates and the resulting apple wines were analysed using headspace-SPME GC-MS. CATA technique with a well-trained panel was applied for sensory evaluation. The results show that the flavour of single-variety apple wine can be significantly altered by taking oenological measures. High correlations were found between the results of the analytical investigation and the sensory evaluation. Maceration of the mash leads to an increase in the fruity character of the products, also reflected by significantly higher fruit ester quantities in the wine. During mash fermentation, spontaneous malolactic fermentation was induced leading to a product with new, but thoroughly interesting sensory properties of the apple wine. The results of this study demonstrate that the integration of oenological measures may open a wide field to the development of a high diversity in apple wine flavour.

Induced effect of Ca2+ and Al3+ on chaetominine synthesis by Aspergillus fumigatus CY018 under submerged fermentation

Chaetominine (CHA), an alkaloid with biological activity produced by Aspergillus fumigatus CY018, has strong anticancer activity against the human leukemia cell. However, its further research for pharmacokinetics is limited by CHA production in submerged fermentation, which is a problem urgently awaiting solutions by effective biological strategies. In this work, Ca2+ and Al3+ exhibited a strong promoting effect on CHA production after multiple metal ions screening. Then, the concentration of Ca2+ and Al3+ and the addition time was optimized. The suitable conditions were 0.5 mM Ca2+ and 0.5 mM Al3+ at 120 h, in which the CHA production reached 58.5 and 49.45 mg/L with optimized condition of Ca2+ and Al3+ respectively. The intermediate metabolites, such as DAHP, chorismate and tryptophan, were increased obviously with addition of Ca2+ and Al3+. The activities of key enzymes of DAHPs, AroAs and TrpCs in CHA biosynthesis pathway were improved by 3.58-, 3.60- and 3.34-folds respectively. Meanwhile, the transcription levels of laeA, dahp, cs, and trpC were upregulated by 3.22-, 12.65-, 5.58- and 6.99-fold respectively by addition of Ca2+ and Al3+. Additionally, the fermentation strategy was successfully scaled up to 5 L reactor, in which CHA production could attain 75.6 mg/L at 336 h. This work demonstrated that Ca2+ and Al3+ co-addition was an effective strategy for increasing CHA production, and the information obtained might be useful to the further improvement of CHA fermentation on large scale.

Cellulosic Ethanol Production by Engineered Consortia of Scheffersomyces Stipitis and Zymomonas Mobilis

Background: As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. Two of the conditions that are needed to attain cost-efficient lignocellulosic ethanol production at an industrial scale are the simultaneous transformation of glucose and xylose to ethanol and a highly efficient ethanol fermentation process. Results: In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, at the end of fermentation compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various concentrations of corn stover hydrolysates, in which the sum of glucose and xylose concentrations in the hydrolysates were 60, 90, and 120 g/L respectively, were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56% - 4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12% - 102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Conclusions: The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. Thus, this strategy has potential for future lignocellulosic ethanol production.

Development of a Combination Fermentation Strategy to Simultaneously Increase Biomass and Enzyme Activity of D-amino acid Oxidase Expressed in Escherichia Coli

d-amino acid oxidase (DAAO) is widely used in the industrial preparation of l-amino acids, and cultivating Escherichia coli (E. coli) expressing DAAO for the biosynthesis of l-phosphinothricin (l-PPT) is very attractive. At present, the biomass production of DAAO by fermentation is still limited in large-scale industrial applications because the expression of DAAO during the fermentation process inhibits the growth of host cells, which limits higher cell density. In this study, the factors that inhibit the growth of bacterial cells during a 5 L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous d-alanine (d-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation. Under optimal conditions, the DO-STAT feeding mode with DO controlled at 30% ± 5% and the addition of 27.5 g/L lactose mixed with 2 g/L d-Ala during induction at 28 °C resulted in the production of 26.03 g dry cell weight (DCW)/L biomass and 390.0 U/g DCW specific activity of DAAO; an increase of 78% and 84%, respectively, compared with the initial fermentation conditions. The fermentation strategy was successfully scale-up to a 5000L fermenter.