Design of a synthetic miniR1 plasmid and its production by engineered Escherichia coli Abstract A synthetic plasmid consisting of the minimal elements for replication control of the R1 replicon and kanamycin resistance marker, which was named pminiR1, was developed. pminiR1 production was tested at 30 °C under aerobic and microaerobic conditions in Escherichia coli W3110 recA− (W1). The plasmid DNA yields from biomass (YpDNA/X) were only 0.06 ± 0.02 and 0.22 ± 0.11 mg/g under aerobic and microaerobic conditions, respectively. As an option to increase YpDNA/X values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The YpDNA/X values using strain W12 increased to 0.85 ± 0.05 and 1.53 ± 0.14 mg/g under aerobic and microaerobic conditions, respectively. pminiR1 production in both strains was compared with that of pUC57Kan at 37 °C under aerobic and microaerobic conditions. The YpDNA/X values for pminiR1 using strain W12 were 6.25 ± 0.16 and 9.27 ± 0.95 mg/g under aerobic and microaerobic conditions, respectively. Such yields were similar to those obtained for plasmid pUC57Kan using strain W12 (6.9 ± 0.64 and 10.85 ± 1.06 mg/g for aerobic and microaerobic cultures, respectively). Therefore, the synthetic minimal plasmid based on the R1 replicon is a valuable alternative to pUC plasmids for biotechnological applications.

Ethanol production from water hyacinth ( Eichhornia crassipes ) hydrolysate by hyper-thermal acid hydrolysis, enzymatic saccharification and yeasts adapted to high concentration of xylose Abstract Water hyacinth (Eichhornia crassipes) was used as a feedstock for ethanol production. The optimal hyper-thermal (HT) acid hydrolysis conditions were 8% (w/v) slurry content, 200 mM H2SO4, at 160 °C for 20 min and enzymatic saccharification for 48 h using an enzyme mixture of 20 units/mL Viscozyme L and Cellic C Tec2. After pretreatment, 48.2 g/L monosaccharides were obtained. Fermentation was conducted with wild and adapted Saccharomyces cerevisiae, Pichia stipitis and Candida lusitaniae. Wild-type S. cerevisiae, P. stipitis, and C. lusitaniae produced 15.3, 19.5 and 22.7 g/L of ethanol, respectively. Adaptive evolution was carried out on 6% (w/v) xylose. S. cerevisiae, P. sipitis and C. lusitaniae adapted to xylose produced 15.3, 21.4 and 23.9 g/L of ethanol with YEtOH of 0.32, 0.44 and 0.49, respectively. These results indicate that water hyacinth has potential as a feed stock for ethanol.

Bioprocess development of a stable FUT8 −/− -CHO cell line to produce defucosylated anti-HER2 antibody Abstract In recent years, an increasing number of defucosylated therapeutic antibodies have been applied in clinical practices due to their better efficacy compared to fucosylated counterparts. The establishment of stable and clonal manufacturing cell lines is the basis of therapeutic antibodies production. Bioprocess development of a new cell line is necessary for its future applications in the biopharmaceutical industry. We engineered a stable cell line expressing defucosylated anti-HER2 antibody based on an established α-1,6-fucosyltransferase (FUT8) gene knockout CHO-S cell line. The optimization of medium and feed was evaluated in a small-scale culture system. Then the optimal medium and feed were scaled up in a bioreactor system. After fed-batch culture over 13 days, we evaluated the cell growth, antibody yield, glycan compositions and bioactivities. The production of anti-HER2 antibody from the FUT8gene knockout CHO-S cells in the bioreactor increased by 37% compared to the shake flask system. The N-glycan profile of the produced antibody was consistent between the bioreactor and shake flask system. The antibody-dependent cellular cytotoxicity activity of the defucosylated antibody increased 14-fold compared to the wild-type antibody, which was the same as our previous results. The results of our bioprocess development demonstrated that the engineered cell line could be developed to a biopharmaceutical industrial cell line.

Kinetic identification of phenolic compounds and potential production of caffeic acid by Ganoderma lipsiense in solid-state fermentation Abstract Basidiomycetes fungi have been utilized for the production of several compounds with bioactive properties, such as phenolic compounds. The present work quantified and identified the phenolic compounds produced in a kinetic study (63 days) and evaluated the antimicrobial activity from the extract obtained by Ganoderma lipsiense cultivation in solid-state fermentation using red rice. Phenolic compounds were identified by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) and caffeic acid content was measured by high-performance liquid chromatography with diode-array detection (HPLC–DAD). Caffeic and syringic acids were produced by G. lipsiense. In the control medium (red rice), the following compounds were identified: p-coumaric acid, salicylic acid, ferulic acid and vanillin. High concentrations of caffeic acid (0.977 µg g−1) were measured in 49 days. Antimicrobial activity was investigated against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus using a minimum inhibitory concentration (MIC) technique. Ganoderma lipsiense extract was only effective against P. aeruginosa. These data have proved to be satisfactory in the study of biosynthesis of caffeic acid and antibacterial compounds by G. lipsiense in solid-state fermentation with red rice.

Valorization of cheese whey and orange molasses for fungal biomass production by submerged fermentation with Rhizopus sp. Abstract Submerged fermentation (SmF) is an attractive biotechnological option for waste treatment, generating fungal bioprotein from food industry by-products. Using different Rhizopus sp. strains as fermentation agents, this paper describes a global strategy to identify interactions between cultivation parameters (pH 4.75–7.75, 7.5–82.5 g/l glucose, 0.75–3.75 g/l nitrogen, incubation time up to 5 days) for valorization of food industry by-products. Selected parameters and/or their interactions are critical for most of the proposed resulting values, giving the opportunity to optimize the process depending on the objective and making an “in silico” pre-evaluation of the process conditions. SmF of orange molasses leads to a high biomass and protein yield (11.9 g biomass/l and 4.0 g protein/l), with 43.1 ± 0.1% of essential amino acids and chemical oxygen demand (COD) reduction of almost 50%. Experiments with cheese whey result in 76.3 ± 1.1% of glucose consumption and a biomass production of 5.6 ± 2.2 g/l with 48.9 ± 5.1% of essential amino acids. SmF of orange molasses and cheese whey could contribute to promoting a sustainable feed industry while reducing by-product treatment. Graphical abstract

Selenium reduction by a defined co-culture of Shigella fergusonii strain TB42616 and Pantoea vagans strain EWB32213-2 Abstract Selenium reduction was evaluated with pure batch cultures of Shigella fergusonii strain TB42616 (TB) and Pantoea vagans strain EWB32213-2 (EWB), respectively. A two-stage process, from Se(VI) to Se(IV) and then from Se(IV) to Se(0), was observed. The second stage of reduction, from Se(IV) to Se(0), was observed as the rate-limiting step resulting in accumulation of the more toxic Se(IV). In order to facilitate Se(VI) reduction and reduce Se(IV) accumulation, the Se(VI)-reducing strain TB was co-cultured with a Se(IV)-reducing strain EWB. Although Se(VI) reduction rate was not affected, Se(IV) reduction was significantly enhanced with low Se(IV) accumulation in the defined co-culture. Effects of culture composition as well as nitrate and arsenate on Se(VI) reduction were also investigated. A co-culture composition of 10:1 (EWB:TB) ratio was observed to achieve the best total selenium reduction. In addition, nitrate at 50 mg/L was observed to inhibit Se(IV) reduction but not Se(VI) reduction, while arsenate at 200 mg/L exhibited slight inhibition on both Se(VI) and Se(IV) reduction.

Performance of an air-cathode microbial fuel cell under varied relative humidity conditions in the cathode chamber Abstract The performance of an air-cathode microbial fuel cell (MFC) with a cap arrangement was significantly affected by humidity conditions in the cathode. An MFC at a relative humidity (RH) of 88% produced a highest cell voltage of 0.42 V (600 Ω) compared to other operations at 50% (0.34 V) and 30% (0.29 V) RHs. During polarization analysis, MFC operation at 88% RH produced a maximum power density of 0.377 W/m2 (a current density of 1.5 A/m2), which was 1.8 and 2.9 times higher than with 50% and 30% RHs, respectively. Cyclic voltammogram analysis revealed a higher reduction current of − 0.073 A with 88% RH. Furthermore, no increase in dissolved oxygen concentration in the anode chamber was observed with 88% RH. This result suggests that control of humidity conditions in cathode chamber could maximize power generation from an air-cathode MFC.

Soybean processing wastewater supported the removal of propyzamide and biochemical accumulation from wastewater by Rhodopseudomonas capsulata Abstract Simultaneous (SPW and propyzamide) wastewater treatment and the production of biochemicals by Rhodopseudomonas capsulata (R. capsulata) were investigated with supplement of soybean processing wastewater (SPW). Compared to control group, propyzamide was removed and biochemicals production were enhanced with the supplement of SPW. Propyzamide induced camH gene expression through activating MAPKKKs gene in MAPK signal transduction pathway. The induction of camH gene and CamH occurs after 1 day for R. capsulata. However, lack of organics in original wastewater did not maintain R. capsulata growth for over 1 day. The supplement of SPW provided sufficient carbon source for R. capsulata under three addition dosages. This new method resulted in the mixed (SPW and propyzamide) wastewater treatment and improvement of biochemicals simultaneously, as well as the realization of reutilization of wastewater and R. capsulata as sludge. Meanwhile, high-order nonlinear mathematical model of the relationship between propyzamide removal rate, Xt and Xt/r, was established.

Product recovery of an enzymatically synthesized (−)-menthol ester in a deep eutectic solvent Abstract Deep eutectic solvents (DESs) have gained increased attention as alternative reaction media for biocatalysis in recent years. There are many investigations on biotransformations in a variety of DESs, but the purification of bioproducts from DES reaction mixtures has not yet been sufficiently addressed. The present study demonstrates a product recovery strategy from a DES reaction medium composed of (−)-menthol and dodecanoic acid. Since the DES is not formed by equimolar amounts of the substrates, but the eutectic point occurs at a 3:1 molar ratio, product isolation is an important task for effective biocatalytic process development, even if the limiting substrate is converted completely. Both DES compounds acted as substrates and reaction solvent in the lipase-catalyzed esterification to synthesize (−)-menthyl dodecanoate. The product (−)-menthyl dodecanoate ester was separated from the DES reaction mixture by a vacuum distillation step and a second esterification reaction can be performed with the recovered (−)-menthol.

Simultaneous removal of Cd 2+ , NO 3 -N and hardness by the bacterium Acinetobacter sp. CN86 in aerobic conditions Abstract This study investigated the factors influencing the simultaneous removal of Cd2+, NO3-N and hardness from water by the bacterial strain CN86. Optimum conditions were determined experimentally by varying the type of organic matter used, initial Cd2+ concentration, and pH. Under the optimum conditions, the maximum removal ratios of Cd2+, NO3-N and hardness were 100.00, 89.85 and 71.63%, respectively. The mechanism of Cd2+ removal is a combination of co-precipitation with calcium carbonate and pH. Further confirmation that Cd2+ can be removed by strain CN86 was provided by XRD and XPS analyses. Meteorological chromatography analysis showed that N2 was produced as an end product. These results demonstrate that the bacterial strain CN86 is a suitable candidate for simultaneously removing Cd2+, NO3-N, and hardness during in wastewater treatment.