a b s t r a c tPervaporation is claimed to be a promising separation technique for the purification of ethanol from fermentation broths during bio-ethanol production. In this study, influence of fermentation by-products on the purification of ethanol from water during hydrophobic pervaporation was investigated.Sugars and salts were found to increase the membrane performance. Reason for this was a change in vapor/liquid equilibrium. 2,3-Butanediol decreased the ethanol flux and selectivity factor, while glycerol exhibited no effect. This was explained by a strong sorption of butanediol into PDMS and no sorption of glycerol. Due to the presence of carboxylic acids, hydrophobicity degree of the Pervap 4060 membrane decreased, which resulted in an irreversible increase in water flux and decrease in separation performance. These observations suggested the presence of silicalite-based fillers in the membrane. When the pH was raised to a value above the dissociation constant, no changes in hydrophobicity degree and membrane performance were found.

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Biocatalytic hydrocarbon oxyfunctionalizations are typically accomplished using oxygenases in living bacteria as biocatalysts. These processes are often limited by either oxygen mass transfer, cofactor regeneration, and/or enzyme instabilities due to the formation of reactive oxygen species. Here, we discuss an alternative approach based on molybdenum (Mo)-containing dehydrogenases, which produce, rather than consume, reducing equivalents in the course of substrate hydroxylation and use water as the oxygen donor. Mo-containing dehydrogenases have a high potential for overcoming limitations encountered with oxygenases. In order to evaluate the suitability and efficiency of a Mo-containing dehydrogenase-based biocatalyst, we investigated quinaldine 4-oxidase (Qox)-containing Pseudomonas strains and the conversion of quinaldine to 4-hydroxyquinaldine. Host strain and carbon source selection proved to be crucial factors influencing biocatalyst efficiency. Resting P. putida KT2440 (pKP1) cells, grown on and induced with benzoate, showed the highest Qox activity and were used for process development. To circumvent substrate and product toxicity/inhibition, a two-liquid phase approach was chosen. Without active aeration and with 1-dodecanol as organic carrier solvent a productivity of 0.4 g l (tot) (-1) h(-1) was achieved, leading to the accumulation of 2.1 g l (tot) (-1) 4-hydroxyquinaldine in 6 h. The process efficiency compares well with values reported for academic and industrially applied biocatalytic oxyfunctionalization processes emphasizing the potential and feasibility of the Qox-containing recombinant cells for heteroaromatic carbon oxyfunctionalizations without the necessity for active aeration.

Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool on targeted genome-editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial strain. A collection of vectors compatible with high-throughput fragment exchange (FX) cloning for heterologous expression in E. coli and Bacillus were previously developed. This vector catalogue was through this work supplemented with editing plasmid for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to a stable integration of the gene into the amyE locus of a seven-protease deficient KO7 strain. The newly engineered B. subtilis strain allowed for a successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering and contributes to a cloning platform for quick transition from HTP enzyme expression to production through fermentation of the industrially relevant B. subtilis and related strains. Importance We complemented a cloning platform with new editing plasmids that allows a quick transition from high-throughput cloning and expression of new enzymes to stable integration of genes for production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate, and by introducing stable expression of subtilisin. The tools should be useful to engineered B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry relevant host.

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In ethanol fermentations, about 2% of the ethanol leaves the fermenter with the offgas. Conventionally, this is recovered by absorption in water. As alternative, vapour permeation was investigated conceptually for ethanol recovery from fermentation off-gas. A preliminary techno-economic evaluation of this system using hydrophobic membrane was carried out. The results were compared with conventional absorption. For the assumed membrane, concentrated ethanol (~ 66 mass%) might be achieved using vapour permeation whereas absorption achieves 2 mass%, and needs much more distillation to achieve ~ 93 mass%. The ethanol recovery costs for base case absorption and for hydrophobic vapour permeation were calculated to be 0.211 and 1.389 US $/kg, respectively. The ethanol recovery cost decreases with increase in membrane permeability in hydrophobic vapour permeation but the base case cost was not achieved. In the vapour permeation process, membrane cost dominates at lower membrane permeabilities whereas at the permeabilities 3 times higher than original, the costs for vacuum on permeate side of membrane governs the ethanol recovery cost.

Animal waste fats were explored as a fermentation substrate for the production of high-value unsaturated single cell oil (SCO) using oleaginous fungi, Mucor circinelloides and Mortierella alpina. Both strains showed good growth and lipid accumulation when using animal fat as a single carbon source. The biomass concentration of 16.7 ± 2.2 gDCW/L and lipid content of 54.1%wt (of dry cell weight) were obtained for Mucor circinelloides in shake flask experiments, surpassing the biomass yield achieved in batch and fed-batch fermentation. In contrast, Mortierella alpina gave the highest biomass concentration (8.3 ± 0.3 gDCW/L) and lipid content (55.8%wt) in fed-batch fermentation. Fat grown Mortierella alpina was able to produce arachidonic acid (ARA), and the highest ARA content of 23.8%wt (of total lipid weight) was in fed-batch fermentation. Gamma-linolenic acid (GLA) was produced by both fungal strains. At the end of fed-batch fermentation, the GLA yields obtained for Mucor circinelloides and Mortierella alpina were 4.51%wt and 2.77%wt (of total lipid weight), respectively. This study demonstrates the production of unsaturated SCO-rich fungal biomass from animal fat by fermentation.