Improvement and scale-down of a Trichoderma reesei shake flask protocol to microtiter plates enables high-throughput screening

Giese, H.; Kruithof, Paulien; Meier, Klaus; Sieben, Michaela; Antonov, Elena; Hommes, R. W. J.

doi:10.1016/j.jbiosc.2014.05.016

Cited by 14 publications

(13 citation statements)

References 38 publications

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“…6), and then stabilized at 5.2 afterwards. Decrease of pH during T. reesei fermentation was previously reported in many other papers [36,37], and this may result from the fact that the optimal pH for growth and protein production of T. reesei was 4 and 5, respectively [38]. Conclusions may be drawn that before the induction for protein expression, there would be a preliminary mycelia growth stage (lasting about 24 h), during which the reducing sugar and pH of fermentation broth would decline.…”

Section: Lipase Production By T Reesei Transformantssupporting

confidence: 50%

Heterologous Expression of an Alkali and Thermotolerant Lipase from Talaromyces thermophilus in Trichoderma reesei

Zhang

Xia

2015

Appl Biochem Biotechnol

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To heterologously express a Talaromyces thermophilus lipase gene in Trichoderma reesei, an efficient binary vector pChph-pCBH1sigpro-ttl which includes a newly designed cbh1 promoter and hygromycin-resistant marker was constructed. This plasmid was then transformed into T. reesei via improved Agrobacterium EHA 105-mediated transformation. After modification of co-culture conditions and enzymolysis treatment of conidia, 258 transformants were produced. A two-step screening method based on antibiotic resistance and capacity to utilize lactose and tributyrin was introduced to further select promising candidates, which would be additionally verified by PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and lipase activity assay. Lipase production was carried out in shaking flasks, and the activity reached 241 IU/mL (7415.4 IU/mg) after 84-h fermentation. It was found that this lipase performed high alkali and thermostable tolerance with the optimal pH 9.5 and temperature 60 °C, and it could retain more than 70 % activity after being disposed in pH 11 or 70 °C for 1 h. This study herein would benefit the genetic engineering of T. reesei and the industrial application of this important fungal lipase.

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Section: Lipase Production By T Reesei Transformantssupporting

confidence: 50%

Heterologous Expression of an Alkali and Thermotolerant Lipase from Talaromyces thermophilus in Trichoderma reesei

Zhang

Xia

2015

Appl Biochem Biotechnol

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“…The suitability of the RAMOS technique also for viscous fermentation broths has been verified [17, 19, 26]. RAMOS flasks, sampling flasks, and flasks for the online power input measurement were filled from the same master mix and cultivated in parallel and under identical conditions to guarantee that cultures ran synchronously.…”

Section: Methodsmentioning

confidence: 99%

Investigation of poly(γ-glutamic acid) production via online determination of viscosity and oxygen transfer rate in shake flasks

et al. 2017

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BackgroundPoly(γ-glutamic acid) (γ-PGA) is a biopolymer with many useful properties making it applicable for instance in food and skin care industries, in wastewater treatment, in biodegradable plastics or in the pharmaceutical industry. γ-PGA is usually produced microbially by different Bacillus spp. The produced γ-PGA increases the viscosity of the fermentation broth. In case of shake flask fermentations, this results in an increase of the volumetric power input. The power input in shake flasks can be determined by measuring the torque of an orbitally rotating lab shaker. The online measurement of the volumetric power input enables to continuously monitor the formation or degradation of viscous products like γ-PGA. Combined with the online measurement of the oxygen transfer rate (OTR), the respiration activity of the organisms can be observed at the same time.ResultsTwo different Bacillus licheniformis strains and three medium compositions were investigated using online volumetric power input and OTR measurements as well as thorough offline analysis. The online volumetric power input measurement clearly depicted changes in γ-PGA formation due to different medium compositions as well as differences in the production behavior of the two investigated strains. A higher citric acid concentration and the addition of trace elements to the standard medium showed a positive influence on γ-PGA production. The online power input signal was used to derive an online viscosity signal which was validated with offline determined viscosity values. The online measurement of the OTR proved to be a valuable tool to follow the respiration activity of the cultivated strains and to determine its reproducibility under different cultivation conditions.ConclusionsThe combination of the volumetric power input and the OTR allows for an easy and reliable investigation of new strains, cultivation conditions and medium compositions for their potential in γ-PGA production. The power input signal and the derived online viscosity directly reflect changes in γ-PGA molecular weight and concentration, respectively, due to different cultivation conditions or production strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-017-0065-4) contains supplementary material, which is available to authorized users.

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“…Eq. was fitted to the data of Giese et al for 24‐well MTPs and is valid for filling volumes of 250–1000 µL/well, shaking frequencies of 150–350 rpm and a constant shaking diameter of 50 mm:

k_{normalL} a,_{24 well} = 20.29 \cdot V_{L,well}^{- 0.74} \cdot n^{1.23} [\frac{1}{normalh}]

…”

Section: Methodsmentioning

confidence: 99%

Permeability of currently available microtiter plate sealing tapes fail to fulfil the requirements for aerobic microbial cultivation

Sieben

Giese

Grosch

et al. 2016

Biotechnology Journal

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Microtiter plate (MTP) sealing tapes are commonly applied in bioprocess development and high throughput screening in order to maintain sterile conditions and avoid liquid evaporation. However, only a few of the commercially available sealing tapes are adequately characterized to guarantee both minimal evaporation and sufficient oxygen supply for aerobic cultivation. Therefore, 12 commercially available sealing tapes are analyzed concerning their water vapor and oxygen permeability. The water vapor permeability is assessed by gravimetrically quantifying the liquid loss due to evaporation. Thereby, the sealing tapes are revealed significant differences. Highly permeable sealing tapes are resulted in liquid loss of up to 25% of the initial filling volume after 8 h at 37°C and 45% ambient humidity. Additionally, the tremendous impact of evaporative cooling on the liquid temperature is detected discovering deviations of up to 3.8°C from the set temperature. The oxygen permeability is assessed by measuring the oxygen transfer rate (OTR). Three out of the 12 tested sealing tapes are impermeable to oxygen while the remaining sealing tapes are ensured sufficient oxygen supply. As a result, all examined sealing tapes are inadequate with respect to either water or oxygen permeation. Based on these novel experimental results, prospective improvements of MTP sealing tapes are presented using a model approach.

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Improvement and scale-down of a Trichoderma reesei shake flask protocol to microtiter plates enables high-throughput screening

Cited by 14 publications

References 38 publications

Heterologous Expression of an Alkali and Thermotolerant Lipase from Talaromyces thermophilus in Trichoderma reesei

Heterologous Expression of an Alkali and Thermotolerant Lipase from Talaromyces thermophilus in Trichoderma reesei

Investigation of poly(γ-glutamic acid) production via online determination of viscosity and oxygen transfer rate in shake flasks

Permeability of currently available microtiter plate sealing tapes fail to fulfil the requirements for aerobic microbial cultivation

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