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New insights into the influence of pre-culture on robust solvent production of C. acetobutylicum
(2024)
Clostridia are known for their solvent production, especially the production of butanol. Concerning the projected depletion of fossil fuels, this is of great interest. The cultivation of clostridia is known to be challenging, and it is difficult to achieve reproducible results and robust processes. However, existing publications usually concentrate on the cultivation conditions of the main culture. In this paper, the influence of cryo-conservation and pre-culture on growth and solvent production in the resulting main cultivation are examined. A protocol was developed that leads to reproducible cultivations of Clostridium acetobutylicum. Detailed investigation of the cell conservation in cryo-cultures ensured reliable cell growth in the pre-culture. Moreover, a reason for the acid crash in the main culture was found, based on the cultivation conditions of the pre-culture. The critical parameter to avoid the acid crash and accomplish the shift to the solventogenesis of clostridia is the metabolic phase in which the cells of the pre-culture were at the time of inoculation of the main culture; this depends on the cultivation time of the pre-culture. Using cells from the exponential growth phase to inoculate the main culture leads to an acid crash. To achieve the solventogenic phase with butanol production, the inoculum should consist of older cells which are in the stationary growth phase. Considering these parameters, which affect the entire cultivation process, reproducible results and reliable solvent production are ensured.
Drought and water shortage are serious problems in many arid and semi-arid regions. This problem is getting worse and even continues in temperate climatic regions due to climate change. To address this problem, the use of biodegradable hydrogels is increasingly important for the application as water-retaining additives in soil. Furthermore, efficient (micro-)nutrient supply can be provided by the use of tailored hydrogels. Biodegradable polyaspartic acid (PASP) hydrogels with different available (1,6-hexamethylene diamine (HMD) and L-lysine (LYS)) and newly developed crosslinkers based on diesters of glycine (GLY) and (di-)ethylene glycol (DEG and EG, respectively) were synthesized and characterized using Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) and regarding their swelling properties (kinetic, absorbency under load (AUL)) as well as biodegradability of PASP hydrogel. Copper (II) and zinc (II), respectively, were loaded as micronutrients in two different approaches: in situ with crosslinking and subsequent loading of prepared hydrogels. The results showed successful syntheses of di-glycine-ester-based crosslinkers. Hydrogels with good water-absorbing properties were formed. Moreover, the developed crosslinking agents in combination with the specific reaction conditions resulted in higher water absorbency with increased crosslinker content used in synthesis (10% vs. 20%). The prepared hydrogels are candidates for water-storing soil additives due to the biodegradability of PASP, which is shown in an exemple. The incorporation of Cu(II) and Zn(II) ions can provide these micronutrients for plant growth.
Perennial ryegrass (Lolium perenne) is an underutilized lignocellulosic biomass that has several benefits such as high availability, renewability, and biomass yield. The grass press-juice obtained from the mechanical pretreatment can be used for the bio-based production of chemicals. Lactic acid is a platform chemical that has attracted consideration due to its broad area of applications. For this reason, the more sustainable production of lactic acid is expected to increase. In this work, lactic acid was produced using complex medium at the bench- and reactor scale, and the results were compared to those obtained using an optimized press-juice medium. Bench-scale fermentations were carried out in a pH-control system and lactic acid production reached approximately 21.84 ± 0.95 g/L in complex medium, and 26.61 ± 1.2 g/L in press-juice medium. In the bioreactor, the production yield was 0.91 ± 0.07 g/g, corresponding to a 1.4-fold increase with respect to the complex medium with fructose. As a comparison to the traditional ensiling process, the ensiling of whole grass fractions of different varieties harvested in summer and autumn was performed. Ensiling showed variations in lactic acid yields, with a yield up to 15.2% dry mass for the late-harvested samples, surpassing typical silage yields of 6–10% dry mass.
Several unconnected laboratory experiments are usually offered for students in instrumental analysis lab. To give the students a more rational overview of the most common instrumental techniques, a new laboratory experiment was developed. Marketed pain relief drugs, familiar consumer products with one to three active components, namely, acetaminophen (paracetamol), acetylsalicylic acid (ASA), and caffeine, were selected. Common analytical methods were compared regarding the performance of qualitative and quantitative analysis of unknown tablets: UV–visible (UV–vis), infrared (IR), and nuclear magnetic resonance (NMR) spectroscopies, as well as high-performance liquid chromatography (HPLC). The students successfully uncovered the composition of formulations, which were divided into three difficulty categories. Students were shown that in addition to simple mixtures handled in theoretical classes, the composition of complex drug products can also be uncovered. By comparing the performance of different techniques, students deepen their understanding and compare the efficiency of analytical methods in the context of complex mixtures. The laboratory experiment can be adjusted for graduate level by including extra tasks such as method optimization, validation, and 2D spectroscopic techniques.
Aspergillus oryzae is an industrially relevant organism for the secretory production of heterologous enzymes, especially amylases. The activities of potential heterologous amylases, however, cannot be quantified directly from the supernatant due to the high background activity of native α-amylase. This activity is caused by the gene products of amyA, amyB, and amyC. In this study, an in vitro CRISPR/Cas9 system was established in A. oryzae to delete these genes simultaneously. First, pyrG of A. oryzae NSAR1 was mutated by exploiting NHEJ to generate a counter-selection marker. Next, all amylase genes were deleted simultaneously by co-transforming a repair template carrying pyrG of Aspergillus nidulans and flanking sequences of amylase gene loci. The rate of obtained triple knock-outs was 47%. We showed that triple knockouts do not retain any amylase activity in the supernatant. The established in vitro CRISPR/Cas9 system was used to achieve sequence-specific knock-in of target genes. The system was intended to incorporate a single copy of the gene of interest into the desired host for the development of screening methods. Therefore, an integration cassette for the heterologous Fpi amylase was designed to specifically target the amyB locus. The site-specific integration rate of the plasmid was 78%, with exceptional additional integrations. Integration frequency was assessed via qPCR and directly correlated with heterologous amylase activity. Hence, we could compare the efficiency between two different signal peptides. In summary, we present a strategy to exploit CRISPR/Cas9 for gene mutation, multiplex knock-out, and the targeted knock-in of an expression cassette in A. oryzae. Our system provides straightforward strain engineering and paves the way for development of fungal screening systems.
This book is based on a multimedia course for biological and chemical engineers, which is designed to trigger students' curiosity and initiative. A solid basic knowledge of thermodynamics and kinetics is necessary for understanding many technical, chemical, and biological processes.
The one-semester basic lecture course was divided into 12 workshops (chapters). Each chapter covers a practically relevant area of physical chemistry and contains the following didactic elements that make this book particularly exciting and understandable:
- Links to Videos at the start of each chapter as preparation for the workshop
- Key terms (in bold) for further research of your own
- Comprehension questions and calculation exercises with solutions as learning checks
- Key illustrations as simple, easy-to-replicate blackboard pictures
Humorous cartoons for each workshop (by Faelis) additionally lighten up the text and facilitate the learning process as a mnemonic. To round out the book, the appendix includes a summary of the most popular experiments in basic physical chemistry courses, as well as suggestions for designing workshops with exhibits, experiments, and "questions of the day."
Suitable for students minoring in chemistry; chemistry majors are sure to find this slimmed-down, didactically valuable book helpful as well. The book is excellent for self-study.
Experimental determination of the cross sections of proton capture on radioactive nuclei is extremely difficult. Therefore, it is of substantial interest for the understanding of the production of the p-nuclei. For the first time, a direct measurement of proton-capture cross sections on stored, radioactive ions became possible in an energy range of interest for nuclear astrophysics. The experiment was performed at the Experimental Storage Ring (ESR) at GSI by making use of a sensitive method to measure (p,γ) and (p,n) reactions in inverse kinematics. These reaction channels are of high relevance for the nucleosyn-thesis processes in supernovae, which are among the most violent explosions in the universe and are not yet well understood. The cross section of the ¹¹⁸Te(p,γ) reaction has been measured at energies of 6 MeV/u and 7 MeV/u. The heavy ions interacted with a hydrogen gas jet target. The radiative recombination process of the fully stripped ¹¹⁸Te ions and electrons from the hydrogen target was used as a luminosity monitor. An overview of the experimental method and preliminary results from the ongoing analysis will be presented.
Preprint: Studies on the enzymatic reduction of levulinic acid using Chiralidon-R and Chiralidon-S
(2023)
The enzymatic reduction of levulinic acid by the chiral catalysts Chiralidon-R and Chiralidon-S which are commercially available superabsorbed alcohol dehydrogenases is described. The Chiralidon®-R/S reduces the levulinic acid to the (R,S)-4-hydroxy valeric acid and the (R)- or (S)- gamma-valerolactone.