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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 preparatio...
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... enable the robot to perform different tasks independently, such as sample homogenization, sample spotting, washing and concentration, we used the concept of modular design for the system development. As shown in Fig. 1, the developed platform is an integration of three modules, namely ultrasonication robot module, centrifuge module and liquid handling module. Each module is able to be operated independently and they can also form a complete system for the full process preparation of fungal samples for FTIR spectroscopy. The machine vision system ...
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... suspension for sample spotting on 384-well IR plates (Bruker Optik GmbH, Germany). In the previous work, we introduced an ultrasonication robot that can provide desired homogeneity of filamentous fungal cell suspension [9]. The robot uses machine vision to screen sample wells and measure the level of fungi homogeneity. In this work, as shown in Fig. 1 and Fig. 7, the ultrasonication robot module was integrated into the sample preparation system for FTIR spectroscopy without hardware modifications. In order to integrate with the other modules, the controller of the ultrasonication robot module (Raspberry Pi 3) was installed with an open-source system Ubuntu MATE to run the software ...
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... shown in Fig. 1, the liquid handling module comprises the right arm (Arm 2) of the dual-arm system, an 8-channel syringe pump (Cavro XMP 6000; TECAN, Switzerland), an electronic pipette (P50; Opentrons, USA), an RGB camera (See3CAM_CU135; e-co systems, USA), a custom-made wash station and a well plate shaker (MicroPlate Genie; Scientific Industries, ...
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... were defined as True Positive (TP) and False Positive (FP), respectively. Undetected objects were marked as False Negative (FN). Then, precision is defined as TP over the sum of TP and FP, while recall is TP over the sum of TP and FN. By varying confidence threshold, the precision-recall curves of the four classes are obtained and shown in Fig. 10. The IoU threshold for the evaluation is the same to the real application (Eq. 1, 0.5). All the four classes show both high precision and recall. High precision and recall represent that most of the objects have been detected and most of the detection results are correct. Further, the average precision of the detection is shown in ...
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... -namely, Mucor circinelloides VI 04473 (Norwegian School of Veterinary Science, Norway) using the same cultivation method as it was described in the previous work [9]. There were 24 wells of samples for each MTP plate, so it created 72 spots on the IR plates. After spotting, the IR plates were dried and scanned to measure the spotting accuracy. Fig. 12 shows the scanned picture of the IR plates and the accuracy measurement method. Generally, the dried samples of fungi on the spots are homogeneous and the spots are located in the center of the well limit circles on the IR plates. As it can be seen in the right enlarged picture, we manually labelled the inner circle of the well limits ...
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... measurement results of two IR plates are shown in Fig. 11a. It can be seen that the positional error test revealed a near normal distribution, indicating that the results seem reliable. Most of the positional errors are located between 0.3 to 0.5 mm, with a mean of 0.36 mm and a 0.15 mm standard deviation. The positional error is mainly caused by the picking up of the pipette tips, because the ...
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... the equation means that the coverage is the overlap area between the blue circle (S blue ) the nearest red circle (S red ) over the red circle (S red ). The coverage rates of two IR plates are shown in Fig. 11b, which indicates that most of the coverage rates are around 0.97 (mean ) with minimum value at 0.81. Our practical experience on the coverage rate suggests a minimum value of 0.8, which means that the system can provide desired samples spots for FTIR analysis. We also recorded the execution time of each procedure for the two tests (one ...
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... at 0.81. Our practical experience on the coverage rate suggests a minimum value of 0.8, which means that the system can provide desired samples spots for FTIR analysis. We also recorded the execution time of each procedure for the two tests (one MTP plate and two MTP plates). The working sequence together with the processing time is displayed in Fig. 13. For two MTP plates (blue blocks), the whole processing time was 942 minutes, during which ultrasonication (c and f) took up most of the time (78.6%) followed by washing of the two MTP plates (12%). In two-MTP mode, the final stage of sample washing (b) and ultrasonication of MTP plate 1 (c) have been processed ...
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... samples using a high-throughput screening spectrometer (HTS-XT; Bruker Optik GmbH, Germany). We extracted the Amide I (using wavenumber of 1650 cm −1 ) absorbance data from the spectra. According to the OPUS Quality Test (OPUS QT) -a standard quality test for FTIR spectra, the absorbance at Amide I band should be in a range 0.3 -1.2. As shown in Fig. 14, 46% of the absorbance in the raw spectra (blue line) is below 0.3. By using the Extended Multiplicative Signal Correction (EMSC) method [22], we can correct the differences in absorbance and obtained the red line. With comparison to the spot coverage rate (green line), we did not find the spot coverage rate has significant influence on the ...
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... enable the robot to perform different tasks independently, such as sample homogenization, sample spotting, washing and concentration, we used the concept of modular design for the system development. As shown in Fig. 1, the developed platform is an integration of three modules, namely ultrasonication robot module, centrifuge module and liquid handling module. Each module is able to be operated independently and they can also form a complete system for the full process preparation of fungal samples for FTIR spectroscopy. The machine vision system ...
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... suspension for sample spotting on 384-well IR plates (Bruker Optik GmbH, Germany). In the previous work, we introduced an ultrasonication robot that can provide desired homogeneity of filamentous fungal cell suspension [9]. The robot uses machine vision to screen sample wells and measure the level of fungi homogeneity. In this work, as shown in Fig. 1 and Fig. 7, the ultrasonication robot module was integrated into the sample preparation system for FTIR spectroscopy without hardware modifications. In order to integrate with the other modules, the controller of the ultrasonication robot module (Raspberry Pi 3) was installed with an open-source system Ubuntu MATE to run the software ...
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... shown in Fig. 1, the liquid handling module comprises the right arm (Arm 2) of the dual-arm system, an 8-channel syringe pump (Cavro XMP 6000; TECAN, Switzerland), an electronic pipette (P50; Opentrons, USA), an RGB camera (See3CAM_CU135; e-co systems, USA), a custom-made wash station and a well plate shaker (MicroPlate Genie; Scientific Industries, ...
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... varying confidence threshold, the precision-recall curves of the four classes are obtained and shown in Fig. 10. The IoU threshold for the evaluation is the same to the real application (Eq. 1, 0.5). All the four classes show both high precision and recall. High precision and recall represent that most of the objects have been detected and most of the detection results are correct. Further, the average precision of the detection is shown in ...
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... -namely, Mucor circinelloides VI 04473 (Norwegian School of Veterinary Science, Norway) using the same cultivation method as it was described in the previous work [9]. There were 24 wells of samples for each MTP plate, so it created 72 spots on the IR plates. After spotting, the IR plates were dried and scanned to measure the spotting accuracy. Fig. 12 shows the scanned picture of the IR plates and the accuracy measurement method. Generally, the dried samples of fungi on the spots are homogeneous and the spots are located in the center of the well limit circles on the IR plates. As it can be seen in the right enlarged picture, we manually labelled the inner circle of the well limits ...
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... The distance between the centroid of the blue circle and the centroid of the nearest red circle relates to the positional error of the pipette tip. To find the nearest red circle, each blue circle was compared to all the red circles and the minimum distance value returns the nearest circle. The measurement results of two IR plates are shown in Fig. 11a. It can be seen that the positional error test revealed a near normal distribution, indicating that the results seem reliable. Most of the positional errors are located between 0.3 to 0.5 mm, with a mean of 0.36 mm and a 0.15 mm standard deviation. The positional error is mainly caused by the picking up of the pipette tips, because the ...
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... the equation means that the coverage is the overlap area between the blue circle (S blue ) the nearest red circle (S red ) over the red circle (S red ). The coverage rates of two IR plates are shown in Fig. 11b, which indicates that most of the coverage rates are around 0.97 (mean ) with minimum value at 0.81. Our practical experience on the coverage rate suggests a minimum value of 0.8, which means that the system can provide desired samples spots for FTIR ...
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... also recorded the execution time of each procedure for the two tests (one MTP plate and two MTP plates). The working sequence together with the processing time is displayed in Fig. 13. For two MTP plates (blue blocks), the whole processing time was 942 minutes, during which ultrasonication (c and f) took up most of the time (78.6%) followed by washing of the two MTP plates (12%). In two-MTP mode, the final stage of sample washing (b) and ultrasonication of MTP plate 1 (c) have been processed simultaneously. The ...
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... samples using a high-throughput screening spectrometer (HTS-XT; Bruker Optik GmbH, Germany). We extracted the Amide I (using wavenumber of 1650 cm −1 ) absorbance data from the spectra. According to the OPUS Quality Test (OPUS QT) -a standard quality test for FTIR spectra, the absorbance at Amide I band should be in a range 0.3 -1.2. As shown in Fig. 14, 46% of the absorbance in the raw spectra (blue line) is below 0.3. By using the Extended Multiplicative Signal Correction (EMSC) method [22], we can correct the differences in absorbance and obtained the red line. With comparison to the spot coverage rate (green line), we did not find the spot coverage rate has significant influence ...
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... FTIR spectroscopy was chosen due to its advantages and wide application in microbiology and biotechnology for the overall biochemical characterization of microbial cells and their metabolites (Carnovale et al., 2021;Forfang et al., 2017;Kohler et al., 2015;Kosa, Shapaval, et al., 2017;Olsen et al., 2023;Shapaval et al., 2017Shapaval et al., , 2023. One of the main advantages of FTIR analysis is that it can be performed on non-destructed or little-processed microbial biomass and combined with automated sample preparation to increase throughput (Li et al., 2016;Xiong et al., 2019). ...
Temperature significantly impacts bacterial physiology, metabolism and cell chemistry. In this study, we analysed lipids and the total cellular biochemical profile of 74 fast‐growing Antarctic bacteria grown at different temperatures. Fatty acid diversity and temperature‐induced alterations aligned with bacterial classification—Gram‐groups, phylum, genus and species. Total lipid content, varied from 4% to 19% of cell dry weight, was genus‐ and species‐specific. Most bacteria increased lipid content at lower temperatures. The effect of temperature on the profile was complex and more species‐specific, while some common for all bacteria responses were recorded. Gram‐negative bacteria adjusted unsaturation and acyl chain length. Gram‐positive bacteria adjusted methyl branching (anteiso‐/iso‐), chain length and unsaturation. Fourier transform infrared spectroscopy analysis revealed Gram‐, genus‐ and species‐specific changes in the total cellular biochemical profile triggered by temperature fluctuations. The most significant temperature‐related alterations detected on all taxonomy levels were recorded for mixed region 1500–900 cm⁻¹, specifically the band at 1083 cm⁻¹ related to phosphodiester groups mainly from phospholipids (for Gram‐negative bacteria) and teichoic/lipoteichoic acids (for Gram‐positive bacteria). Some changes in protein region were detected for a few genera, while the lipid region remained relatively stable despite the temperature fluctuations.
... This would enable use of FTIR spectroscopy as quantitative methodology replacing tedious GC-FID analysis. Finally, FTIR spectroscopy combined with the recently developed automated sample preparation solutions [64][65][66] allow to perform rapid and extraordinary HTS that would bring optimization of PHA production to the advanced level of complexity. ...
High-throughput screening (HTS) methods for characterization of microbial production of polyhydroxyalkanoates (PHA) are currently under investigated, despite the advent of such systems in related fields. In this study, phenotypic microarray by Biolog PM1 screening of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99 identified 49 and 54 carbon substrates to be metabolized by these bacteria, respectively. Growth on 15 (Halomonas sp. R5-57) and 14 (Pseudomonas sp. MR4-99) carbon substrates was subsequently characterized in 96-well plates using medium with low nitrogen concentration. Bacterial cells were then harvested and analyzed for putative PHA production using two different Fourier transform infrared spectroscopy (FTIR) systems. The FTIR spectra obtained from both strains contained carbonyl-ester peaks indicative of PHA production. Strain specific differences in the carbonyl-ester peak wavenumber indicated that the PHA side chain configuration differed between the two strains. Confirmation of short chain length PHA (scl-PHA) accumulation in Halomonas sp. R5-57 and medium chain length PHA (mcl-PHA) in Pseudomonas sp. MR4-99 was done using Gas Chromatography-Flame Ionization Detector (GC-FID) analysis after upscaling to 50 mL cultures supplemented with glycerol and gluconate. The strain specific PHA side chain configurations were also found in FTIR spectra of the 50 mL cultures. This supports the hypothesis that PHA was also produced in the cells cultivated in 96-well plates, and that the HTS approach is suitable for analysis of PHA production in bacteria. However, the carbonyl-ester peaks detected by FTIR are only indicative of PHA production in the small-scale cultures, and appropriate calibration and prediction models based on combining FTIR and GC-FID data needs to be developed and optimized by performing more extensive screenings and multivariate analyses.
... For the dry film approach, the drying step might take a few minutes, and sometimes also a dilution step is needed to avoid non-linear detector responses. However, the approach is highly suited for high-throughput analysis using multi-well transmission plates, and it has been shown that the procedure from sample to dry film formation and subsequent FTIR analysis can be readily automated [36]. Thus, there is a potential for the development of this approach as an automated at-line solution for measurements of protein quality in industrial processes. ...
Whereas industrial laboratory systems for Fourier-Transform Infrared (FTIR) spectroscopic characterization have been available for decades, dedicated FTIR solutions for in-line process control or for industrial at-line analysis of protein quality are still scarce. Thus, the present study aimed to compare two industrially viable sampling techniques, namely attenuated total reflectance (ATR) and dry film FTIR spectroscopy, for qualitative and quantitative analysis of liquid protein solutions. For this purpose, two sample sets were acquired: set 1 consists of 95 protein hydrolysate samples produced in the laboratory from salmon processing by-products, and set 2 consists of 133 protein hydrolysate samples obtained from an industrial processing plant of poultry by-products. Average molecular weights (AMW) and Brix measurements of protein hydrolysates were used as reference values for the calibration of regression models. AMW was predicted with higher accuracy and lower estimation errors using dry film FTIR compared to ATR measurements for both sample types. The difference in predictive performance was higher in poultry hydrolysates because the protein and tissue complexities are higher than in salmon hydrolysates, and information from the amide I region in the FTIR spectra is needed to provide good calibration results. ATR, on the other hand, was more reliable for the prediction of Brix values of protein hydrolysates. The study also showed that FTIR spectroscopy can be used to predict protein quality features of industrially produced protein hydrolysates with a sensitivity of high industrial relevance. Therefore, developing industrially viable portable instruments for at-line process analysis in the food, feed, and biotech industries is a natural next development stage.
... Many studies have focused on mobile robotic systems that handle transportation across distributed workbenches [17], [18]. Several studies have improved workbench tasks using robots, such as pipetting [19] and colony picking [20]. A recent notable achievement was the use of a mobile manipulator to autonomously search for improved photocatalysts to produce hydrogen from water in a process that required only eight days; by contrast, human researchers took several months to complete this task [11]. ...
Grinding materials into a fine powder is a time-consuming task in material science that is generally performed by hand, as current automated grinding machines might not be suitable for preparing small-sized samples. This study presents a robotic powder grinding system for laboratory automation in material science applications that observe the powder's state to improve the grinding outcome. We developed a soft jig consisting of off-the-shelf gel materials and 3D-printed parts, which can be used with any robot arm to perform powder grinding. The jig's physical softness allows for safe grinding without force sensing. In addition, we developed a visual feedback system that observes the powder distribution and decides where to grind and when to gather. The results showed that our system could grind 79 percent of the powder to a particle size smaller than 200 µm by using the soft jig and visual feedback. This ratio was 57% when using only the soft jig without feedback. Our system can be used immediately in laboratories to alleviate the workload of researchers.
... FTIR spectroscopy is a rapid, non-destructive next-generation phenotyping technique widely used in medicine [23][24][25][26][27][28], food industry [29,30], and biotechnology [31][32][33][34] for discrimination, identification, and characterization of different microorganisms [9,33,[35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]. FTIR spectroscopy can be applied in a high-throughput set-up and there are automated sample preparation systems for FTIR available [51][52][53]. Previously, we successfully applied this technique for the biochemical fingerprinting of the same bacterial strains cultivated on broth and agar media and identified a possible accumulation of intracellular polymer polyhydroxyalkanoates (PHA) and lipids for some isolates [9]. In this study we combined FTIR spectroscopy with the multivariate data analysis for detecting temperature and nutrient-induced changes of the chemical phenotype of the Antarctic green snow bacteria. ...
Temperature fluctuations and nutrient composition are the main parameters influencing green snow microbiome. In this study we investigated the influence of temperature and nutrient conditions on the growth and cellular chemical profile of bacteria isolated from green snow. Chemical profiling of the green snow bacteria was done by high-throughput FTIR spectroscopy combined with multivariate data analysis. We showed that temperature and nutrients fluctuations strongly affect growth ability and chemical profile of the green snow bacteria. The size of colonies for green snow bacteria grown at higher (25 °C) and lower (4 °C and 10 °C) than optimal temperature (18 °C) was smaller. All isolates grew on rich medium, and only 19 isolates were able to grow on synthetic minimal media. Lipid and mixed spectral regions showed to be phylogeny related. FTIR fingerprinting indicates that lipids are often affected by the temperature fluctuations. Growth on different media resulted in the change of the whole chemical profile, where lipids showed to be more affected than proteins and polysaccharides. Correlation analysis showed that nutrient composition is clearly strongly influencing chemical changes in the cells, followed by temperature.
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... A real-time dry-film FTIR solution would require robotic sample handling and drying. Such an approach has already been proposed for a high-throughput laboratory screening system (Xiong, Shapaval, Kohler, Li, & From, 2019). Even though adjustments would be needed for an industrial system, this shows the potential of the approach. ...
Enzymatic protein hydrolysis can be used industrially for refining peptides from poultry by-products. The aim is to introduce these peptides for human consumption, but today the majority is used for feed or pet food. One main barrier for entering the human market is unknown and unstable peptide product qualities. Reliable analytical methods for characterising the peptide fraction is therefore vital for optimizing and controlling the product quality.
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We also show that the database can be used for evaluating industrial samples. By comparing the FTIR fingerprints of industrial samples with those in the database, we could see systematic changes in product quality over time and make hypotheses about the underlying causes for change. These results lay the foundation for using dry-film FTIR spectroscopy as an industrial tool for product and process optimisation and control.
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... Since the early 90s, FTIR spectroscopy has been used for rapid identification and classification of microorganisms in the food and pharmaceutical industry (Van de Voort, 1992, Roggo et al., 2007, Shapaval et al., 2010, Shapaval et al., 2012, Shapaval et al., 2013, Lohumi et al., 2015, for epidemiological investigations in medicine (Amiali et al., 2007, Essendoubi et al., 2007, Colabella et al., 2017, for high-throughput screenings of microbial cell factories (Curk et al., 1994, Kohler et al., 2015, Li et al., 2016, Kosa et al., 2017a, Kosa et al., 2017b, Marova et al., 2017, Kosa et al., 2018b, Shapaval et al., 2019, Xiong et al., 2019a, Xiong et al., 2019b and biotechnological applications (Naumann, 2000, Harz et al., 2009. ...
Snow microorganisms play a significant role in climate change and affecting the snow melting rate in the Arctic and Antarctic regions. While research on algae inhabiting green and red snow has been performed extensively, bacteria dwelling in this biotope have been studied to a much lesser extent. In this study, we performed 16S rRNA gene amplicon sequencing of two green snow samples collected from the coastal area of the eastern part of Antarctica and conducted genotypic and phenotypic profiling of 45 fast‐growing bacteria isolated from these samples. 16S rRNA gene amplicon sequencing of two green snow samples showed that bacteria inhabiting these samples are mostly represented by families Burkholderiaceae (46.31%), Flavobacteriaceae (22.98%), and Pseudomonadaceae (17.66%). Identification of 45 fast‐growing bacteria isolated from green snow was performed using 16S rRNA gene sequencing. We demonstrated that they belong to the phyla Actinobacteria and Proteobacteria, and are represented by the genera Arthrobacter, Cryobacterium, Leifsonia, Salinibacterium, Paeniglutamicibacter, Rhodococcus, Polaromonas, Pseudomonas, and Psychrobacter. Nearly all bacterial isolates exhibited various growth temperatures from 4°C to 25°C, and some isolates were characterized by a high level of enzymatic activity. Phenotyping using Fourier transform infrared (FTIR) spectroscopy revealed a possible accumulation of intracellular polymer polyhydroxyalkanoates (PHA) or lipids in some isolates. The bacteria showed different lipids/PHA and protein profiles. It was shown that lipid/PHA and protein spectral regions are the most discriminative for differentiating the isolates.
... [47] The Opentrons is a liquid handler typically used for biological applications such as polymer chain reaction (PCR), [33,34] nucleic acid extraction and purification, DNA assembly, [35] protein preparation [36] as well as fungal sample preparation. [48] It can also be used for making dilutions. And finally, a UV/Vis microplate reader is often/commonly used for biological absorption and fluorescence assays. ...
We have developed a high‐throughput system to synthesise and explore up to 96 heterogeneous catalysts at the same time. The system was developed as a proof of concept, using a standard glass plate and a 3D printed 96‐well plate. Nano‐droplets of catalyst formulations were transferred to the glass plate using an acoustic liquid handler and upon heat treatments, the miniature mesoporous metal oxide (MMO) catalysts were formed. The 3D printed bottomless 96‐well plate was fixed to the glass plate, to give 96 individual wells, each containing a catalyst. Four catalyst plates were prepared (Co3O4‐, Au/Co3O4‐, Pd/Co3O4‐ and Co/Mn‐MMO) to be screened for their activity in the oxidation of morin, as a model reaction. The observed reaction rates (kobs) for each catalyst were calculated to identify the most active catalyst. The general method described herein requires microscopic amounts of catalysts with derivates of the catalyst's composition. A high‐throughput method was developed for the screening of up to 96 mesoporous metal oxide catalysts. This system was created by employing robotics such as an acoustic liquid handler for the miniature catalyst formation, as well as 3D printing. This proof of concept shows that mesoporous metal oxide could be miniaturised by the deposition of nano‐droplet on a glass plate, and screened for their activity in the oxidation of morin as a model reaction.Robotic Catalysis: A High‐Throughput Method for Miniature Screening of Mesoporous Metal Oxides (Meijboom) @go2uj @Prof_Meijboom
