A Fully Automated Robot for the Preparation of Fungal Samples for FTIR Spectroscopy Using Deep Learning

Manual preparation of fungal samples for Fourier Transform Infrared (FTIR) spectroscopy involves sample washing, homogenization, concentration and spotting, which requires time-consuming and repetitive operations, making it unsuitable for screening studies. This paper presents the design and development of a fully automated robot for the preparation of fungal samples for FTIR spectroscopy. The whole system was constructed based on a previously-developed ultrasonication robot module, by adding a newly-designed centrifuge module and a newly-developed liquid handling module. The liquid handling module consists of a high accuracy electric pipette for spotting and a low accuracy syringe pump for sample washing and concentration. A dual robotic arm system with a gripper connects all of the hardware components. Furthermore, a camera on the liquid handling module uses deep learning to identify the labware settings, which includes the number and positions of well plates and pipette tips. Machine vision on the ultrasonication robot module can detect the sample wells and return the locations to the liquid handling module, which makes the system hand-free for users. Tight integration of all the modules enables the robot to process up to two 96-well microtiter (MTP) plates of samples simultaneously. Performance evaluation shows the deep learning based approach can detect four classes of labware with high average precision, from 0.93 to 1.0. In addition, tests of all procedures show that the robot is able to provide homogeneous sample spots for FTIR spectroscopy with high positional accuracy and spot coverage rate

Oleaginous yeasts respond differently to carbon sources present in lignocellulose hydrolysate

Background Microbial oils, generated from lignocellulosic material, have great potential as renewable and sustainable alternatives to fossil-based fuels and chemicals. By unravelling the diversity of lipid accumulation physiology in different oleaginous yeasts grown on the various carbon sources present in lignocellulose hydrolysate (LH), new targets for optimisation of lipid accumulation can be identified. Monitoring lipid formation over time is essential for understanding lipid accumulation physiology. This study investigated lipid accumulation in a variety of oleaginous ascomycetous and basidiomycetous strains grown in glucose and xylose and followed lipid formation kinetics of selected strains in wheat straw hydrolysate (WSH). Results Twenty-nine oleaginous yeast strains were tested for their ability to utilise glucose and xylose, the main sugars present in WSH. Evaluation of sugar consumption and lipid accumulation revealed marked differences in xylose utilisation capacity between the yeast strains, even between those belonging to the same species. Five different promising strains, belonging to the species Lipomyces starkeyi, Rhodotorula glutinis, Rhodotorula babjevae and Rhodotorula toruloides, were grown on undiluted wheat straw hydrolysate and lipid accumulation was followed over time, using Fourier transform-infrared (FTIR) spectroscopy. All five strains were able to grow on undiluted WSH and to accumulate lipids, but to different extents and with different productivities. R. babjevae DVBPG 8058 was the best-performing strain, accumulating 64.8% of cell dry weight (CDW) as lipids. It reached a culture density of 28 g/L CDW in batch cultivation, resulting in a lipid content of 18.1 g/L and yield of 0.24 g lipids per g carbon source. This strain formed lipids from the major carbon sources in hydrolysate, glucose, acetate and xylose. R. glutinis CBS 2367 also consumed these carbon sources, but when assimilating xylose it consumed intracellular lipids simultaneously. Rhodotorula strains contained a higher proportion of polyunsaturated fatty acids than the two tested Lipomyces starkeyi strains. Conclusions There is considerable metabolic diversity among oleaginous yeasts, even between closely related species and strains, especially when converting xylose to biomass and lipids. Monitoring the kinetics of lipid accumulation and identifying the molecular basis of this diversity are keys to selecting suitable strains for high lipid production from lignocellulose

Montana Kaimin, January 30, 2008

Student newspaper of the University of Montana, Missoula.https://scholarworks.umt.edu/studentnewspaper/6138/thumbnail.jp

Assessment of genetically modified maize Bt11 x MIR162 x MIR604 x MON 89034 x 5307 x GA21 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 (application EFSA-GMO-DE-2018-149)

Bt11 x MIR162 x MIR604 x MON 89034 x 5307 x GA21 was produced by conventional breeding of the GM maize events Bt11, MIR162, MIR604, MON 89034, 5307 and GA21. Accordingly, Bt11 x MIR162 x MIR604 x MON 89034 x 5307 x GA21 maize produces the transgenic proteins in the individual GM maize events (Cry1Ab, PAT, Vip3Aa20, PMI, mCry3A, MIR604 PMI, Cry1A.105, Cry2Ab2, eCry3.1Ab and mEPSPS). Event Bt11 maize expresses the insecticidal protein Cry1Ab that protects against feeding damage caused by certain lepidopteran pests and the phosphinothricin acetyltransferase (PAT) protein for weed control by providing tolerance to herbicide products containing glufosinate ammonium. Event MIR162 maize expresses the insecticidal protein Vip3Aa20 that protects against feeding damage caused by certain lepidopteran pests and the PMI protein which enables transformed plant cells to utilise mannose as a primary carbon source and therefore used as a selectable marker in the development of the MIR162 maize. Event MIR604 maize expresses the insecticidal protein mCry3A that protects against feeding damage caused by certain coleopteran pests and the MIR604 PMI protein which enables transformed plant cells to utilise mannose as a primary carbon source and therefore used as a selectable marker in the development of the MIR604 maize. Event MON 89034 maize expresses the insecticidal proteins Cry1A.105 and Cry2Ab2 that protect against feeding damage caused by certain lepidopteran pests. Event 5307 maize expresses the insecticidal protein eCry3.1Ab that protects against feeding damage caused by certain coleopteran pests and the PMI protein which enables transformed plant cells to utilise mannose as a primary carbon source and therefore used as a selectable marker in the development of the 5307 maize. Event GA21 expresses the double-mutated 5-enolpyruvylshikimate-3-phosphate synthase enzyme (mEPSPS) for weed control by providing tolerance to herbicide products containing glyphosate.The scientific documentation provided in the application for genetically modified maize Bt11 x MIR162 x MIR604 x MON 89034 x 5307 x GA21 is adequate for risk assessment, and in accordance with EFSA guidance on risk assessment of genetically modified plants for use in food or feed. The VKM GMO panel does not consider the introduced modifications in maize Bt11 x MIR162 x MIR604 x MON 89034 x 5307 x GA21 to imply potential specific health or environmental risks in Norway, compared to EU-countries The EFSA opinion is adequate also for Norwegian considerations. Therefore, a full risk assessment of maize Bt11 x MIR162 x MIR604 x MON 89034 x 5307 x GA21 was not performed by the VKM GMO Panel.Assessment of genetically modified maize Bt11 x MIR162 x MIR604 x MON 89034 x 5307 x GA21 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 (application EFSA-GMO-DE-2018-149)publishedVersio

Rhodotorula kratochvilovae CCY 20-2-26-The Source of Multifunctional Metabolites

Multifunctional biomass is able to provide more than one valuable product, and thus, it is attractive in the field of microbial biotechnology due to its economic feasibility. Carotenogenic yeasts are effective microbial factories for the biosynthesis of a broad spectrum of biomolecules that can be used in the food and feed industry and the pharmaceutical industry, as well as a source of biofuels. In the study, we examined the effect of different nitrogen sources, carbon sources and CN ratios on the co-production of intracellular lipids, carotenoids, beta-glucans and extracellular glycolipids. Yeast strain R. kratochvilovae CCY 20-2-26 was identified as the best co-producer of lipids (66.7 +/- 1.5% of DCW), exoglycolipids (2.42 +/- 0.08 g/L), beta-glucan (11.33 +/- 1.34% of DCW) and carotenoids (1.35 +/- 0.11 mg/g), with a biomass content of 15.2 +/- 0.8 g/L, by using the synthetic medium with potassium nitrate and mannose as a carbon source. It was shown that an increased C/N ratio positively affected the biomass yield and production of lipids and beta-glucans

Study of Metabolic Adaptation of Red Yeasts to Waste Animal Fat Substrate

Carotenogenic yeasts are non-conventional oleaginous microorganisms capable to utilize various waste substrates. In this work 4 red yeast strains (Rhodotorula, Cystofilobasidium and Sporobolomyces sp.) were cultivated in media containing crude, emulsified and enzymatically hydrolysed animal waste fat, compared with glucose and glycerol as single C-sources. Cell morphology (cryo-SEM, TEM), production of biomass, lipase, biosurfactants, lipids (GC/FID) carotenoids, ubiquinone, ergosterol (HPLC/PDA) in yeast cells was studied depending on medium composition, C-source and C/N ratio. All studied strains are able to utilize solid and processed fat. Biomass production at C/N=13 was higher on emulsified/hydrolysed fat than on glucose/glycerol. Production of lipids and lipidic metabolites was enhanced for several times on fat; the highest yields of carotenoids (24.8 mg/l) and lipids (54.5%/CDW) were found in S.pararoseus. Simultaneous induction of lipase and biosurfactants was observed on crude fat substrate. Increased C/N ratio (13-100) led to higher biomass production in fat media. Production of total lipids increased in all strains to C/N 50. Oppositely, production of carotenoids, ubiquinone and ergosterol dramatically decreased with increased C/N in all strains. Compounds accumulated in stressed red yeasts are having great application potential and can result from valorization of animal waste fat in the biorefinery concept.Karotenogenní kvasinky jsou schopny utilizovat řadu různých substrátů včetně odpadů. V této práci je popsána kultivace vybraných kmenů karotenogenních kvasinek rodů Rhodotorula, Cystofilobasidium, and Sporobolomyces sp. v médiu obsahujícícm surový odpadní živočišný tuk a upravený substrát emulsifikací a enzymovou hydrolýzou. Výsledky byly srovnány s kultivací na glukóze a glycerolu jako jednoduchých zdrojích uhlíku

Raman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganisms

Background Monitoring and control of both growth media and microbial biomass is extremely important for the development of economical bioprocesses. Unfortunately, process monitoring is still dependent on a limited number of standard parameters (pH, temperature, gasses etc.), while the critical process parameters, such as biomass, product and substrate concentrations, are rarely assessable in-line. Bioprocess optimization and monitoring will greatly benefit from advanced spectroscopy-based sensors that enable real-time monitoring and control. Here, Fourier transform (FT) Raman spectroscopy measurement via flow cell in a recirculatory loop, in combination with predictive data modeling, was assessed as a fast, low-cost, and highly sensitive process analytical technology (PAT) system for online monitoring of critical process parameters. To show the general applicability of the method, submerged fermentation was monitored using two different oleaginous and carotenogenic microorganisms grown on two different carbon substrates: glucose fermentation by yeast Rhodotorula toruloides and glycerol fermentation by marine thraustochytrid Schizochytrium sp. Additionally, the online FT-Raman spectroscopy approach was compared with two at-line spectroscopic methods, namely FT-Raman and FT-infrared spectroscopies in high throughput screening (HTS) setups. Results The system can provide real-time concentration data on carbon substrate (glucose and glycerol) utilization, and production of biomass, carotenoid pigments, and lipids (triglycerides and free fatty acids). Robust multivariate regression models were developed and showed high level of correlation between the online FT-Raman spectral data and reference measurements, with coefficients of determination (R2) in the 0.94–0.99 and 0.89–0.99 range for all concentration parameters of Rhodotorula and Schizochytrium fermentation, respectively. The online FT-Raman spectroscopy approach was superior to the at-line methods since the obtained information was more comprehensive, timely and provided more precise concentration profiles. Conclusions The FT-Raman spectroscopy system with a flow measurement cell in a recirculatory loop, in combination with prediction models, can simultaneously provide real-time concentration data on carbon substrate utilization, and production of biomass, carotenoid pigments, and lipids. This data enables monitoring of dynamic behaviour of oleaginous and carotenogenic microorganisms, and thus can provide critical process parameters for process optimization and control. Overall, this study demonstrated the feasibility of using FT-Raman spectroscopy for online monitoring of fermentation processes.Raman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganismspublishedVersio

Assessment of genetically modified maize MON 95379 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2020‐170)

Event MON 95379 is a genetically modified maize developed by a two-step process. In the first step, immature embryos of maize inbred line LH244 were co-cultured with a disarmed Agrobacterium tumefaciens (also known as Rhizobium radiobacter) strain ABI containing the vector PV-ZMIR522223. In the second step, selected R2 lines were crossed with maize inbred LH244 line expressing Crerecombinase, which had been transformed with vector PVZMOO513642. In the resulting plants, the CP4 EPSPS-cassette (used for selection of transformed plants) was excised by the Cre recombinase, and the Cre gene was subsequently segregated away, through conventional breeding, to obtain maize MON 95379. Maize MON 95379 expresses Cry1B.868, a chimeric protein containing domains from Cry1A, Cry1B and Cry1C naturally expressed in Bacillus thuringiensis, and Cry1Da_7, an optimised version of Cry1Da carrying four amino acids substitutions to increase its activity. The two Cry proteins expressed in maize MON 95379 provide protection against targeted pests within the order of butterflies and moths (Lepidoptera) including fall armyworm (Spodoptera frugiperda), sugarcane borer (Diatraea saccharalis) and corn earworm (Helicoverpa zea). The scientific documentation provided in the application for genetically modified maize MON 95379 is adequate for risk assessment, and in accordance with EFSA guidance on risk assessment of genetically modified plants for use in food or feed. The VKM GMO panel does not consider the introduced modifications in event MON 95379 to imply potential specific health or environmental risks in Norway, compared to EU-countries. The EFSA opinion is adequate also for Norwegian considerations. Therefore, a full risk assessment of event MON 95379 was not performed by the VKM GMO Panel.Assessment of genetically modified maize MON 95379 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2020‐170)publishedVersio

Assessment of genetically modified maize DP41143 x MON890343 x MON 874113 x DAS40278-9 and sub-combinations for food and feed uses, import and processing under Regulation (EC) No 1829/2003 (application EFSA GMO-NL-2020-171)

Genetically modified maize DP41149 x MON 890349 x MON 874119 x DAS-40278-9 was developed by crossing to combine four single events: DP4114, MON 89034, MON 87411 and DAS-40278-9. DP4114 express the Cry1F protein to confer protection against certain lepidopteran pests, the Cry34Ab1 and Cry35Ab1 proteins to confer protection against certain coleopteran pests and PAT protein to confer tolerance to glufosinate-ammonium-containing herbicides. MON 89034 express the Cry1A.105 and Cry2Ab2 proteins to confer protection against certain lepidopteran pests. MON 87411 express the Cry3Bb1 protein to confer protection against certain coleopteran larvae and the DvSnf7 dsRNA confer protection against western corn rootworm, and the CP4 EPSPS protein for tolerance to glyphosate containing herbicides. DAS-40278-9 express the AAD-1 protein to catalyse the degradation of the general class ofherbicides known as aryloxyphenoxypropionates (AOPP) and to confer tolerance to 2,4- dichlorophenoxyacetic acid (2,4-D) herbicides.Assessment of genetically modified maize DP41143 x MON890343 x MON 874113 x DAS40278-9 and sub-combinations for food and feed uses, import and processing under Regulation (EC) No 1829/2003 (application EFSA GMO-NL-2020-171)publishedVersionpublishedVersio