Microtiter plate cultivation of oleaginous fungi and monitoring of lipogenesis by high-throughput FTIR spectroscopy.

BACKGROUND: Oleaginous fungi can accumulate lipids by utilizing a wide range of waste substrates. They are an important source for the industrial production of omega-6 polyunsaturated fatty acids (gamma-linolenic and arachidonic acid) and have been suggested as an alternative route for biodiesel production. Initial research steps for various applications include the screening of fungi in order to find efficient fungal producers with desired fatty acid composition. Traditional cultivation methods (shake flask) and lipid analysis (extraction-gas chromatography) are not applicable for large-scale screening due to their low throughput and time-consuming analysis. Here we present a microcultivation system combined with high-throughput Fourier transform infrared (FTIR) spectroscopy for efficient screening of oleaginous fungi. RESULTS: The microcultivation system enables highly reproducible fungal fermentations throughout 12 days of cultivation. Reproducibility was validated by FTIR and HPLC data. Analysis of FTIR spectral ester carbonyl peaks of fungal biomass offered a reliable high-throughput at-line method to monitor lipid accumulation. Partial least square regression between gas chromatography fatty acid data and corresponding FTIR spectral data was used to set up calibration models for the prediction of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, unsaturation index, total lipid content and main individual fatty acids. High coefficients of determination (R2 = 0.86-0.96) and satisfactory residual predictive deviation of cross-validation (RPDCV = 2.6-5.1) values demonstrated the goodness of these models. CONCLUSIONS: We have demonstrated in this study, that the presented microcultivation system combined with rapid, high-throughput FTIR spectroscopy is a suitable screening platform for oleaginous fungi. Sample preparation for FTIR measurements can be automated to further increase throughput of the system.

A portable dry film FTIR instrument for industrial food and bioprocess applications.

The main objective of this study was to design, build, and test a compact, multi-well, portable dry film FTIR system for industrial food and bioprocess applications. The system features dry film sampling on a circular rotating disc comprising 31 wells, a design that was chosen to simplify potential automation and robotic sample handling at a later stage. Calibration models for average molecular weight (AMW, 200 samples) and collagen content (68 samples) were developed from the measurements of industrially produced protein hydrolysate samples in a controlled laboratory environment. Similarly, calibration models for the prediction of lactate content in samples from cultivation media (59 samples) were also developed. The portable dry film FTIR system showed reliable model characteristics which were benchmarked with a benchtop FTIR system. Subsequently, the portable dry film FTIR system was deployed in a bioprocessing plant, and protein hydrolysate samples were measured at-line in an industrial environment. This industrial testing involved building a calibration model for predicting AMW using 60 protein hydrolysate samples measured at-line using the portable dry film FTIR system and subsequent model validation using a test set of 26 samples. The industrial calibration in terms of coefficient of determination (R2 = 0.94), root mean square of cross-validation (RMSECV = 194 g mol-1), and root mean square of prediction (RMSEP = 162 g mol-1) demonstrated low prediction errors as compared to benchtop FTIR measurements, with no statistical difference between the calibration models of the two FTIR systems. This is to the authors' knowledge the first study for developing and employing a portable dry film FTIR system in the enzymatic protein hydrolysis industry for successful at-line measurements of protein hydrolysate samples. The study therefore suggests that the portable dry film FTIR instrument has huge potential for in/at-line applications in the food and bioprocessing industries.

Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 days.

Introduction: Successful long-term expansion of skeletal muscle satellite cells (MuSCs) on a large scale is fundamental for cultivating animal cells for protein production. Prerequisites for efficient cell expansion include maintaining essential native cell activities such as cell adhesion, migration, proliferation, and differentiation while ensuring consistent reproducibility. Method: This study investigated the growth of bovine MuSC culture using low-volume spinner flasks and a benchtop stirred-tank bioreactor (STR). Results and discussion: Our results showed for the first time the expansion of primary MuSCs for 38 days in a bench-top STR run with low initial seeding density and FBS reduction, supported by increased expression of the satellite cell marker PAX7 and reduced expression of differentiation-inducing genes like MYOG, even without adding p38-MAPK inhibitors. Moreover, the cells retained their ability to proliferate, migrate, and differentiate after enzymatic dissociation from the microcarriers. We also showed reproducible results in a separate biological benchtop STR run.

Feed microbiome: confounding factor affecting fish gut microbiome studies.

There is an increasing interest in the impact of feed on the fish gut microbiome. Most of the studies are based on sequencing the bacterial housekeeping gene 16S rRNA from extracted total DNA, including resident and non-resident live bacteria as well as dead bacteria. It has not been a common practice to include the feed as control, although it contains various nutritious ingredients that microorganisms can use before or after feed preparation. Thus, study designs using digesta as a proxy for the intestinal microbiome raise the concern that composition of the gut microbiome might be biased by carry-over of microbial DNA from the feed itself. Here we report analysis of 15 feeds and representative intestinal digesta of Atlantic salmon (Salmo salar) from five independent case studies. This allowed us to identify "feed microbiomes" that were microbially diverse and shared taxa with digesta microbiomes. Digesta-specific microbiomes were identified, though they were mainly enriched by a few taxa, such as Mycoplasma and Ruminococcaceae. Overall, findings are consistent with a model wherein gut microbial profiles are to a different degree influenced by bacterial DNA present in the feed itself through a "feed microbiome" carry-over effect.