Fermentation in 24-well plates is an efficient screening platform for filamentous fungi

Linde, T. C. de K. van der; Hansen, Niels Ebbe; Lübeck, Mette; Lübeck, Peter Stephensen

doi:10.1111/lam.12268

Cited by 25 publications

(26 citation statements)

References 23 publications

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“…Fermentation setup in 24-well plates was made following the principles of Linde et al [28] and citric acid production medium from [38] …”

Section: Fermentation Setup and Conditionsmentioning

confidence: 99%

“…When LaeA was deleted in A. carbonarius the production of ochratoxin, a potential human carcinogen, was decreased with 68-97 % compared to wild type and the production of conidia in light and in dark was decreased as well [9]. A. carbonarius is closely related to A. niger [1] and similarly to A. niger it produces citric acid in high amounts when cultivated at optimal conditions [28,49].…”

Section: Introductionmentioning

confidence: 95%

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The global regulator LaeA controls production of citric acid and endoglucanases inAspergillus carbonarius

Linde

Zoglowek

Lübeck

et al. 2016

Journal of Industrial Microbiology and Biotechnology

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The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74-96 % compared to the wild type. The endoglucanase activity was reduced with 51-78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.

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“…Fermentation setup in 24-well plates was made following the principles of Linde et al [28] and citric acid production medium from [38] …”

Section: Fermentation Setup and Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

The global regulator LaeA controls production of citric acid and endoglucanases inAspergillus carbonarius

Linde

Zoglowek

Lübeck

et al. 2016

Journal of Industrial Microbiology and Biotechnology

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“…When a large number of strains or cultivation conditions have to be tested, a high throughput screening (HTS) system is required, which can yield reproducible and scalable results. In order to increase throughput compared to traditional shake flasks, miniaturization of cultivations by microtiter plates or microbioreactors is desired [3–5]. HTS of filamentous fungi in microtiter plates is a challenging task because (a) there is a substantial risk of cross-contamination between individual cultivations, (b) highly viscous fermentation broth can cause oxygen transfer limitation and local inhomogeneity, and (c) excessive wall growth may take place, which favors sporulation.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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BackgroundOleaginous 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.ResultsThe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0716-7) contains supplementary material, which is available to authorized users.

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“…Galactaric acid production by T. reesei has previously been demonstrated in flask cultures [8], but at much lower concentrations than can be obtained in bioreactors [7]. Running and Bansal [12] demonstrated that 24-well plates can have as good or better oxygen transfer as shaken flasks, depending on the shaking regime applied, and 24-well plates are increasingly being used for the cultivation of filamentous fungi [13–15]. It is therefore useful to assess whether galactaric acid production by T. reesei could be scaled down for production in 24-well plates using pectin as substrate.…”

Section: Introductionmentioning

confidence: 99%

Scaling up and scaling down the production of galactaric acid from pectin using Trichoderma reesei

2017

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BackgroundBioconversion of d-galacturonic acid to galactaric (mucic) acid has previously been carried out in small scale (50–1000 mL) cultures, which produce tens of grams of galactaric acid. To obtain larger amounts of biologically produced galactaric acid, the process needed to be scaled up using a readily available technical substrate. Food grade pectin was selected as a readily available source of d-galacturonic acid for conversion to galactaric acid.ResultsWe demonstrated that the process using Trichoderma reesei QM6a Δgar1 udh can be scaled up from 1 L to 10 and 250 L, replacing pure d-galacturonic acid with commercially available pectin. T. reesei produced 18 g L−1 galactaric acid from food-grade pectin (yield 1.00 g [g d-galacturonate consumed]−1) when grown at 1 L scale, 21 g L−1 galactaric acid (yield 1.11 g [g d-galacturonate consumed]−1) when grown at 10 L scale and 14 g L−1 galactaric acid (yield 0.77 g [g d-galacturonate consumed]−1) when grown at 250 L scale. Initial production rates were similar to those observed in 500 mL cultures with pure d-galacturonate as substrate. Approximately 2.8 kg galactaric acid was precipitated from the 250 L culture, representing a recovery of 77% of the galactaric acid in the supernatant. In addition to scaling up, we also demonstrated that the process could be scaled down to 4 mL for screening of production strains in 24-well plate format. Production of galactaric acid from pectin was assessed for three strains expressing uronate dehydrogenase under alternative promoters and up to 11 g L−1 galactaric acid were produced in the batch process.ConclusionsThe process of producing galactaric acid by bioconversion with T. reesei was demonstrated to be equally efficient using pectin as it was with d-galacturonic acid. The 24-well plate batch process will be useful screening new constructs, but cannot replace process optimisation in bioreactors. Scaling up to 250 L demonstrated good reproducibility with the smaller scale but there was a loss in yield at 250 L which indicated that total biomass extraction and more efficient DSP would both be needed for a large scale process.

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Fermentation in 24-well plates is an efficient screening platform for filamentous fungi

Cited by 25 publications

References 23 publications

The global regulator LaeA controls production of citric acid and endoglucanases inAspergillus carbonarius

The global regulator LaeA controls production of citric acid and endoglucanases inAspergillus carbonarius

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

Scaling up and scaling down the production of galactaric acid from pectin using Trichoderma reesei

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