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This study compared the effects of white and red thyme oil against natural biofilm. Thyme oil contains various volatile biologically active substances that can affect the growth of biofilm (algae, cyanobacteria, bacteria). In the first step, white thyme oil was therefore tested in flasks using a plastic lid (air-tight) or a cotton plug (air-permeable). The aim was to determine whether the method of closing the test containers affects the amount of biomass obtained. This was measured using a spectrometer at a wavelength of 750 nm. The results showed a clear positive effect of the cotton plug on the growth of the biofilm without a significant effect on the inhibition rate of the tested oil concentration. In the next step, the algicidal effectiveness of white and red thyme oil (10 mg/L and 100 mg/L) against biofilm was compared. The results showed a higher efficiency of red oil. Therefore, red thyme oil was selected for further research.

Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L-1 of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L-1 CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production.Key points• Evaluation of shake flask designs for cultivating with hydrophobic raw materials• Development of a workflow for microwell plate cultivations with hydrophobic raw materials• Production of polyhydroxyalkanoate in small scale experiments from waste animal fat

Lycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures.Key messageScaling-up of Lycium schweinfurthii suspension culture is possible in a rocking motion bioreactor for production of phenolics and lupeol.

The chemistry literature contains more than a century's worth of instructions for making molecules, all written by and for humans. Steiner et al. developed an autonomous compiler and robotic laboratory platform to synthesize organic compounds on the basis of standardized methods descriptions (see the Perspective by Milo). The platform comprises conventional equipment such as round-bottom flasks, separatory funnels, and a rotary evaporator to maximize its compatibility with extant literature. The authors showcase the system with short syntheses of three common pharmaceuticals that proceeded comparably to manual synthesis. Science , this issue p. eaav2211 ; see also p. 122 A compiler directs a robotic platform to conduct short organic syntheses using standard protocols and laboratory equipment. The synthesis of complex organic compounds is largely a manual process that is often incompletely documented. To address these shortcomings, we developed an abstraction that maps commonly reported methodological instructions into discrete steps amenable to automation. These unit operations were implemented in a modular robotic platform by using a chemical programming language that formalizes and controls the assembly of the molecules. We validated the concept by directing the automated system to synthesize three pharmaceutical compounds, diphenhydramine hydrochloride, rufinamide, and sildenafil, without any human intervention. Yields and purities of products and intermediates were comparable to or better than those achieved manually. The syntheses are captured as digital code that can be published, versioned, and transferred flexibly between platforms with no modification, thereby greatly enhancing reproducibility and reliable access to complex molecules.

Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae . After the successful in vitro production of S. jeffreyense -inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml −1 after 14 days, in comparison to 75% and 99,875 IJs ml −1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml −1 ), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo.

Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties. The Pichia pastoris expression system of LHyal that depends on AOX1 promoter (PAOX1) has been constructed. However, the addition of the toxic inducer methanol is a big safety concern. Here, a combinational strategy was adopted for constitutive expression of LHyal to high level in P. pastoris. By optimizing the combination of promoters PGAP, PGAP(m), and PTEF1 and signal peptides α-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 × 105 U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 × 105 U/mL. In addition, the impact of overexpressing transcription factors Aft1, Gal4-like, and Yap1 on LHyal production was also investigated. We found the co-expression of Aft1 significantly enhanced the expression of LHyal to 3.03 × 105 U/mL. Finally, LHyal activity of 2.12 × 106 U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 × 104 U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.

5-Hydroxytryptophan (5-HTP) is the precursor of the neurotransmitter serotonin and has been used for the treatment of various diseases such as depression, insomnia, chronic headaches, and binge eating associated obesity. The production of 5-HTP had been achieved in our previous report, by the development of a recombinant strain containing two plasmids for biosynthesis of L-tryptophan (L-trp) and subsequent hydroxylation. In this study, the L-trp biosynthetic pathway was further integrated into the E. coli genome, and the promoter strength of 3-deoxy-7-phosphoheptulonate synthase, which catalyzes the first step of L-trp biosynthesis, was engineered to increase the production of L-trp. Hence, the 5-HTP production could be manipulated by the regulation of copy number of L-trp hydroxylation plasmid. Finally, the 5-HTP production was increased to 1.61 g/L in the shaking flasks, which was 24% improvement comparing to the original producing strain, while the content of residual L-trp was successfully reduced from 1.66 to 0.2 g/L, which is beneficial for the downstream separation and purification. Our work shall promote feasible progresses for the industrial production of 5-HTP.

Driven by the concept of plug-and-play cell culture-based viral vaccine production using disposable bioreactors, we evaluated an orbital shaken bioreactor (OSB) for human influenza A virus production at high cell concentration. Therefore, the OSB model SB10-X was coupled to two hollow fiber-based perfusion systems, namely, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). The AGE1.CR.pIX avian suspension cells grew to 50 × 106 cells/mL in chemically defined medium, maintaining high cell viabilities with an average specific growth rate of 0.020 h−1 (doubling time = 32 h). Maximum virus titers in the range of 3.28–3.73 log10(HA units/100 µL) were achieved, corresponding to cell-specific virus yields of 1000–3500 virions/cell and productivities of 0.5–2.2 × 1012 virions/L/d. This clearly demonstrates the potential of OSB operation in perfusion mode, as results achieved in a reference OSB batch cultivation were 2.64 log10(HA units/100 µL), 1286 virions/cell and 1.4 × 1012 virions/L/d, respectively. In summary, the SB10-X bioreactor can be operated with ATF and TFF systems, which is to our knowledge the first report regarding OSB operation in perfusion mode. Moreover, the results showed that the system is a promising cultivation system for influenza A virus vaccine production. The OSB disposable bioreactor has the potential for simplifying the scale-up from shake flasks to the large-scale bioreactor, facilitating rapid responses in the event of epidemics or pandemics.

A live-attenuated, human vaccine against mosquito-borne yellow fever virus has been available since the 1930s. The vaccine provides long-lasting immunity and consistent mass vaccination campaigns counter viral spread. However, traditional egg-based vaccine manufacturing requires about 12 months and vaccine supplies are chronically close to shortages. In particular, for urban outbreaks, vaccine demand can be covered rarely by global stockpiling. Thus, there is an urgent need for an improved vaccine production platform, ideally transferable to other flaviviruses including Zika virus. Here, we present a proof-of-concept study regarding cell culture-based yellow fever virus 17D (YFV) and wild-type Zika virus (ZIKV) production using duck embryo-derived EB66® cells. Based on comprehensive studies in shake flasks, 1-L bioreactor systems were operated with scalable hollow fiber-based tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) perfusion systems for process intensification. EB66® cells grew in chemically defined medium to cell concentrations of 1.6 × 108 cells/mL. Infection studies with EB66®-adapted virus led to maximum YFV titers of 7.3 × 108 PFU/mL, which corresponds to about 10 million vaccine doses for the bioreactor harvest. For ZIKV, titers of 1.0 × 1010 PFU/mL were achieved. Processes were automated successfully using a capacitance probe to control perfusion rates based on on-line measured cell concentrations. The use of cryo-bags for direct inoculation of production bioreactors facilitates pre-culture preparation contributing to improved process robustness. In conclusion, this platform is a powerful option for next generation cell culture-based flavivirus vaccine manufacturing.