The monitoring of oil production process by deep learning based on morphology in oleaginous yeasts.

BACKGROUND: Monitoring jar fermenter-cultured microorganisms in real time is important for controlling productivity of bioproducts in large-scale cultivation settings. Morphological data is used to understand the growth and fermentation states of these microorganisms during monitoring. Oleaginous yeasts are used for their high productivity of single-cell oils but the relationship between lipid productivity and morphology has not been elucidated in these organisms. RESULTS: In this study, we investigated the relationship between the morphology of oleaginous yeasts (Lipomyces starkeyi and Rhodosporidium toruloides were used) and their cultivation state in a large-scale cultivation setting using a real-time monitoring system. We combined this with deep learning by feeding a large amount of high-definition cell images obtained from the monitoring system to a deep learning algorithm. Our results showed that the cell images could be grouped into 7 distinct groups and that a strong correlation existed between each group and its biochemical activity (growth and oil-productivity). CONCLUSIONS: This is the first report describing the morphological variations of oleaginous yeasts in a large-scale cultivation, and describes a promising new avenue for improving productivity of microorganisms in large-scale cultivation through the use of a real-time monitoring system combined with deep learning. KEY POINTS: • A real-time monitoring system followed the morphological change of oleaginous yeasts. • Deep learning grouped them into 7 distinct groups based on their morphology. • A correlation between the cultivation state and the shape of the yeast was observed.

A real-time monitoring system for automatic morphology analysis of yeast cultivation in a jar fermenter.

The monitoring of microbial cultivation in real time and controlling their cultivation aid in increasing the production yield of useful material in a jar fermenter. Common sensors such as dissolved oxygen (DO) and pH can easily provide general-purpose indexes but do not reveal the physiological states of microbes because of the complexity of measuring them in culture conditions. It is well known from microscopic observations that the microbial morphology changes in response to the intracellular state or extracellular environment. Recently, studies have focused on rapid and quantitative image analysis techniques using machine learning or deep learning for gleaning insights into the morphological, physiological or gene expression information in microbes. During image analysis, it is necessary to retrieve high-definition images to analyze the microbial morphology in detail. In this study, we have developed a microfluidic device with a high-speed camera for the microscopic observation of yeast, and have constructed a system capable of generating their morphological information in real-time and at high definition. This system was connected to a jar fermenter, which enabled the automatic sampling for monitoring the cultivation. We successfully acquired high-definition images of over 10,000 yeast cells in about 2.2 s during ethanol fermentation automatically for over 168 h. We recorded 33,600 captures containing over 1,680,000 cell images. By analyzing these images, the morphological changes of yeast cells through ethanol fermentation could be captured, suggesting the expansion of the application of this system in controlling microbial fermentation using the morphological information generated. KEY POINTS: • Enables real-time visualization of microbes in a jar fermenter using microscopy. • Microfluidic device for acquiring high-definition images. • Generates a large amount of image data by using a high-speed camera.

Large refractive index changes of a chemically amplified photoresist in femtosecond laser nonlinear lithography.

We found that marked increases in refractive index of chemically amplified photoresists induced by highly repetitive femtosecond laser irradiation without post-exposure baking treatment. For laser writing speed less than 30 µm/s, the refractive index change of the nonlinear absorption region was as large as 8 × 10(-3). Moreover, cross-linking reactions of the resists were induced. The refractive index changes can generate optical confinement and subsequent channel propagations of femtosecond laser pulses. The coupling efficiency was estimated as high as 87% using a low numerical aperture objective lens. The peak intensities of the guiding modes exceeded the polymerization threshold of the resist.

Cu Patterning Using Femtosecond Laser Reductive Sintering of CuO Nanoparticles under Inert Gas Injection.

In this paper, we report the effect of inert gas injection on Cu patterning generated by femtosecond laser reductive sintering of CuO nanoparticles (NPs). Femtosecond laser reductive sintering for metal patterning has been restricted to metal and metal-oxide composite materials. By irradiating CuO-nanoparticle paste with femtosecond laser pulses under inert gas injection, we intended to reduce the generation of metal oxides in the formed patterns. In an experimental evaluation, the X-ray diffraction peaks corresponding to copper oxides, such as CuO and Cu2O, were much smaller under N2 and Ar gas injections than under air injection. Increasing the injection rates of both gases increased the reduction degree of the X-ray diffraction peaks of the CuO NPs, but excessively high injection rates (≥100 mL/min) significantly decreased the surface density of the patterns. These results qualitatively agreed with the ratio of sintered/melted area. The femtosecond laser reductive sintering under inert gas injection achieved a vacuum-free direct writing of metal patterns.

Direct Writing of Cu Patterns on Polydimethylsiloxane Substrates Using Femtosecond Laser Pulse-Induced Reduction of Glyoxylic Acid Copper Complex.

We investigate the direct writing properties of copper (Cu) patterns on glass and polydimethylsiloxane (PDMS) substrates using femtosecond laser pulse-induced thermochemical reduction of glyoxylic acid copper (GACu) complex. The films of the GACu complex coated on the substrates were irradiated by focused femtosecond laser pulses using a low numerical aperture of 0.45. Under the same conditions, such as laser scanning speed and pulse energy, the width of the line patterns fabricated on PDMS substrates was larger than that on glass substrates. X-ray diffraction peaks of the patterns on glass substrates corresponded to Cu without significant oxidation. By contrast, although Cu patterns were fabricated on PDMS substrates at a scanning speed of 10 mm/s and pulse energy of 0.49 nJ, Cu2O was also generated under overheating conditions at a scanning speed of 1 mm/s and pulse energy of 0.37 nJ. All the patterns exhibited electrical conductivity. The minimum resistivity of the patterns on PDMS substrates is 1.4 × 10-5 Ωm, which is 10 times higher than that on glass substrates, indicating that microcracks formed by thermal shrinkage of the substrates during the laser irradiation increase the resistivity. This direct Cu writing technique on soft materials is useful for fabricating flexible microdevices.

Effect of Substrates on Femtosecond Laser Pulse-Induced Reductive Sintering of Cobalt Oxide Nanoparticles.

Direct writing of cobalt/cobalt oxide composites has attracted attention for its potential use in catalysts and detectors in microsensors. In this study, cobalt-based composite patterns were selectively formed on glass, polyethylene naphthalate (PEN), and polyethylene terephthalate (PET) substrates via the femtosecond laser reductive sintering of Co3O4 nanoparticles in an ambient atmosphere. A Co3O4 nanoparticle ink, including the nanoparticles, ethylene glycol as a reductant, and polyvinylpyrrolidone as a dispersant, was spin-coated onto the substrates. Near-infrared femtosecond laser pulses were then focused and scanned across the ink films to form the patterns. The non-sintered nanoparticles were subsequently removed from the substrate. The resulting sintered patterns were found to be made up of Co/CoO composites on the glass substrates, utilizing various pulse energies and scanning speeds, and the Co/CoO/Co3O4 composites were fabricated on both the PEN and PET substrates. These results suggest that the polymer substrates with low thermal resistance react with the ink during the reductive sintering process and oxidize the patterns more easily compared with the patterns on the glass substrates. Such a direct writing technique of cobalt/cobalt oxide composites is useful for the spatially selective printing of catalysts and detectors in functional microsensors.

Copper and Nickel Microsensors Produced by Selective Laser Reductive Sintering for Non-Enzymatic Glucose Detection.

In this work, the method of selective laser reductive sintering was used to fabricate the sensor-active copper and nickel microstructures on the surface of glass-ceramics suitable for non-enzymatic detection of glucose. The calculated sensitivities for these microsensors are 1110 and 2080 µA mM-1·cm-2 for copper and nickel, respectively. Linear regime of enzymeless glucose sensing is provided between 0.003 and 3 mM for copper and between 0.01 and 3 mM for nickel. Limits of glucose detection for these manufactured micropatterns are equal to 0.91 and 2.1 µM for copper and nickel, respectively. In addition, the fabricated materials demonstrate rather good selectivity, long-term stability and reproducibility.

Direct Writing of Copper Micropatterns Using Near-Infrared Femtosecond Laser-Pulse-Induced Reduction of Glyoxylic Acid Copper Complex.

We have fabricated Cu-based micropatterns in an ambient environment using femtosecond laser direct writing to reduce a glyoxylic acid Cu complex spin-coated onto a glass substrate. To do this, we scanned a train of focused femtosecond laser pulses over the complex film in air, following which the non-irradiated complex was removed by rinsing the substrates with ethanol. A minimum line width of 6.1 µm was obtained at a laser-pulse energy of 0.156 nJ and scanning speeds of 500 and 1000 µm/s. This line width is significantly smaller than that obtained in previous work using a CO2 laser. In addition, the lines are electrically conducting. However, the minimum resistivity of the line pattern was 2.43 × 10-6 Ω·m, which is ~10 times greater than that of the pattern formed using the CO2 laser. An X-ray diffraction analysis suggests that the balance between reduction and re-oxidation of the glyoxylic acid Cu complex determines the nature of the highly reduced Cu patterns in the ambient air.

SiO2-based nonplanar structures fabricated using femtosecond laser lithography.

SiO2-based hybrid diffractive-refractive microlenses were fabricated by femtosecond laser lithography-assisted micromachining, which is a combined process of nonlinear lithography and plasma etching. The high-aspect-ratio patterns of resist were formed by laser exposure without translating the laser spot. By scanning this rod three-dimensionally, micro-Fresnel lens patterns were written directly inside resists on the convex lenses. Then, the resist patterns were transferred to the underlying lenses by CHF(3) plasma. We obtained SiO2 nonplanar structures with smooth surfaces. This hybridization shifted the focal length of the lens by 216 microm, which was consistent with theoretical value.

Effect of Heat Accumulation on Femtosecond Laser Reductive Sintering of Mixed CuO/NiO Nanoparticles.

Direct laser-writing techniques have attracted attention for their use in two- and three-dimensional printing technologies. In this article, we report on a micropatterning process that uses femtosecond laser reductive sintering of mixed CuO/NiO nanoparticles. The writing speed, laser fluence, and incident total energy were varied to investigate the influence of heat accumulation on the micropatterns formed by these materials. Heat accumulation and the thermal history of the laser irradiation process significantly affected the material composition and the thermoelectric properties of the fabricated micropatterns. Short laser irradiation durations and high laser fluences decrease the amount of metal oxide in the micropatterns. Selective fabrication of p-type and n-type thermoelectric micropatterns was demonstrated to be possible with control of the reduction and reoxidization reactions through the control of writing speed and total irradiation energy.