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Conventionally oxygen is considering as an inhibitor of methanogenesis process. However, a studies showed that in case of anaerobic digestion process the oxygen could improve the efficiency of the process. According to that results the impact of microaeration on ex – situ hydrogenotrophic methanogenesis was tested. As a result, it was observed a microorganism, belonging to Methanobacteriales order show a good tolerance for oxygen. The process moreover is not inhibited by load of oxygen equal to 0.45% in relation to injected hydrogen. The results showed a promising future for electrolysis powered with renewable energy sources, which sources are fluctuating and often depends on natural conditions, which force process to be ran in start – stop mode. The potential oxygen contamination could be tolerated by microorganisms according to obtained results. It is opening a new way to develop electrolysis and hydrogen production process.
This study explores the development of a quantitative PCR (qPCR) method for measuring fungal biomass in mixed fermentation of Aspergillus oryzae strains grown on soy okara. The research aimed to address the challenge of differentiating between fungal and plant biomass during the bioconversion of food industry by-products. Key steps included optimizing biomass harvesting techniques, developing a selective qPCR assay, and establishing a correlation between qPCR output and dry mycelial biomass.
A 100 µm strainer method was identified for reliable biomass harvesting. The qPCR assay development involved in-silico and in-vitro primer analysis, with the ITS5 and 58A2R primer pair selected. Despite challenges with high GC content affecting the melting curve, the assay was optimized for selectivity and sensitivity.
Correlation between qPCR copy number and dry mycelial biomass was established for A. oryzae WK and KS strains, with R² values of 0.9795 and 0.9326, respectively. This enabled the quantification of mycelial biomass throughout the fermentation process, revealing that after 48 hours, mycelial biomass accounted for 24.92% and 31.95% of the total mixed biomass for White Koji and Koji Shoyu strains, respectively.
Developed method provides a tool for monitoring fungal growth in mixed biomass systems, supporting research in sustainable food production and the valorization of food industry by-products through fungal fermentation.
The objective of this research was to optimise and validate the use of biowaste as a growth medium for Trichoderma reesei RL-P37, aiming to produce proteins, particularly cellulases, in a cost-effective and sustainable manner. The study consisted of four fermentation experiments conducted using various biowaste compositions, ranging from partial (60%) to complete (100%) substitution of synthetic components with biowaste. A standard laboratory medium was used as a reference to assess protein productivity and biomass yield. In addition, a preliminary T. reesei growth in shaking flasks experiment was carried out to evaluate the suitability of biowaste as a growth medium, confirming that biowaste could support the growth of T. reesei and even resulted in slightly higher biomass production compared to the laboratory medium. Despite similar final protein concentrations across all fermentations (approximately 4 g/L), variations in productivity were observed, with the laboratory medium demonstrating the highest efficiency, followed by 60% and 100% biowaste-based media, respectively. Analysis of yield coefficients revealed that biowaste can sustain growth and protein production; however, its effectiveness was influenced by lot variability and potential inhibitory compounds. Additionally, nutrient imbalances and the presence of possible inhibitory substances posed challenges, as observed in the differences between batch and fed-batch phases. This research demonstrates the potential of biowaste as a complete medium for industrial fermentations, provided nutrient imbalances and inhibitory effects are addressed through optimisation. Further refinement in biowaste preparation and supplementation is required to ensure consistent results and maximise cost-effectiveness for T. reesei fermentations.
Gluconobacter oxydans, an acetic acid bacterium with distinct oxidative metabolic pathways, offers significant industrial potential due to its ability to produce valuable chemicals such as Lsorbose, dihydroxyacetone and gluconic acid. This work aims to advance genetic engineering techniques for G. oxydans, with focus on the development and optimization of regulatable gene expression systems. We examine the evolution from constitutive to inducible gene expression systems in G. oxydans and highlight the adaptation of the L-arabinose-dependent AraC-ParaBAD and L-rhamnose-dependent RhaS-PrhaBAD systems from Escherichia coli. The study identifies specific challenges in these systems, including high inducer concentrations and medium acidification for AraC-ParaBAD, and regulatory anomalies for RhaS-PrhaBAD, such as unexpected expression by L-rhamnose. Additionally, we introduce novel photocaged inducers, 6-Nitropiperonyl-β-L-arabinopyranoside and 6-Nitropiperonyl-β-L-rhamnopyranoside, for lightcontrolled gene expression, despite encountering issues with solubility and UV-induced effects.
The study further also includes the construction of integrative plasmids for the targeted genomic restoration of the key genes gdhM and sldAB in G. oxydans BP.9, and the genomic integration of PGOX0384-rhaT in G. oxydans 621H Δupp. The study includes a detailed analysis of reporter gene expression modulation using both conventional and caged inducers. These findings contribute to the advancement of genetic tools for G. oxydans, offering a pathway to more efficient and sustainable biotechnological applications.
Metalloendopeptidases belong to a class of enzymes that digest proteins dependently on a divalent metal ion as a cofactor, which is usually zinc. Metalloendopeptidases can be specific for the N-terminus or C-terminus of different amino acids. For instance, the Peptidyl-Lys class is specific for the amino acid lysine and various peptidases were previously isolated from the fruiting bodies of fungi, such as Grifola frondosa, Pleurotus ostreatus, and Armillaria mellea (Zhao et al., 2020).
The previously discovered metalloendopeptidases specific for lysine were active at an optimum alkaline pH, however the recently isolated Tc-LysN is an endopeptidase which has a pH optimum in acidic conditions. Tc-LysN is a peptidyl-lys metalloendopeptidase, an endopeptidase specific for the N-terminus of lysine. Its resistance to high temperature and its activity optimum at acidic pH (Ahmed et al., 2024) makes it a valuable option for proteomic experiments. The peptidase used in this Master Thesis was genetically engineered in P. pastoris by Uzair et al.
Initially, the P. pastoris mutant was cultivated in shake flasks with different growing conditions, looking for an increased titer of the protein. Afterward, the protein was produced and isolated with a single step anion-exchange chromatography at pH 7.2.
After the peptidase was purified, its apoenzyme was originated using the chelating agent 1,10-phenantroline, which successfully inhibited the enzyme. Subsequently, the apoenzyme was reactivated with Zn2+ and Co2+. The reactivated apoenzymes achieved diverse biochemical characteristics, compared to the native enzyme, presenting an increased enzyme activity of 110% for the Co2+ reactivated apoenzyme, although paired with less specificity. Furthermore, also a reduction of temperature resistance was recorded, where after 1 hour of incubation at 70 °C less than 10% activity was recorded.
Finally, the studies on this kind of peptidases are important because of their possible pivotal role in proteomic experiments (Zhao et al., 2020). The modification of the biochemical characteristics of these peptidases could give important tools that could be used in future applications.
The growing threat posed by multidrug-resistant (MDR) pathogens, such as Klebsiella pneumoniae (Kp), represents a significant challenge in modern medicine. Traditional antibiotic therapies are often ineffective against these pathogens, leading to high mortality rates. MDR Kp infections pose a novel challenge in military medical contexts, particularly in Medical Biodefense, as they can be deliberately spread, leading to resource-intensive care in military centres. Recognizing this issue, the European Defence Agency initiated a prioritised research project in 2023 (EDF Resilience PHAGE- SGA 2023). To address this challenge, the Bundeswehr Institute of Microbiology (IMB) leads BMBF- (Federal Ministry of Education and Research) and EU-funded projects on the use of bacteriophages as adjuvant therapy alongside antibiotics. Since 2017, the IMB has isolated and characterised Kp phages, collecting over 600 isolates and optimizing their production for therapy, in compliance with the EMA (European Medicine Agency) guidelines. This involves in vitro phage genome packaging to minimize endotoxin load, reduce manufacturing costs, and shorten production times. The goal of this work was to establish MinION sequencing (Oxford Nanopore Technology) as a quick and reliable way for initial identification and characterisation of phage genomes. Especially as a quick screening method for isolated on Kp, prior to more precise but also more expensive and time consuming sequencing methods like Illumina. This characterisation is crucial for developing a personalized pipeline aimed at producing magistral or Good Manufacturing Practice (GMP) quality medicinal phage solutions tailored individually for each patient. DNA extraction methods were compared to identify suitable input DNA for sequencing purposes. Additionally, the quality of this DNA was as- sessed to determine its suitability for in vitro phage packaging, which was successfully done achieving a phage titer of 103, confirming that the DNA used for MinION sequencing could indeed be used for acellular packaging. The created genomes were annotated and compared with Illumina sequencing, revealing high similarity in all five individually tested cases. Between the generated sequences only a 4% maximal percentual difference in genome size was observed, while simultaneously showing high similarity in the actual sequence. Throughout the course of this study, a total of 645.15 GB of sequencing data were generated. In total, 38 phages were successfully characterised, with 21 phage genomes assembled and annotated, and saved in the IMB database.
Self-sufficient enzymes belong to the cytochrome P450 (CYP) group and are known for their superior hydroxylation catalytic activity. In the pursuit of identifying new pesticides to combat antimicrobial-resistant pathogens, we employed BM3 wild type (BM3-WT), the fastest monohydroxylating CYP, along with its seven homologs, to investigate the production of potential hydroxylated derivatives from the established pesticide, 4-oxocrotonic acid using high-pressure liquid chromatography (HPLC) method. Following the recombinant production of BM3-WT and three other homologs in E. coli, and their subsequent purification using Immobilized Metal Affinity Chromatography (IMAC), a novel enzyme assay approach was developed as a substitute for the carbon monoxide (CO) assay. This new method relied on the measurement of NADPH consumption at 340 nm by BM3-WT for palmitic acid. Leveraging this established technique, we explored the substrate specificity of BM3-WT and its homologs not only on palmitic acid but also on other structurally similar compounds, including 4-oxocrotonic acid. The results obtained from the established NADPH assay indicate that all tested enzymes displayed greater catalytic activity on 4-oxocrotonic acid in comparison to other substrates with similar structures. However, the impact of BM3-WT and its homologs on 4-oxocrotonic acid varied in terms of product specificity. Enzymes such as Poh, Trr and Bas-CYP D exhibited specificity in producing solely monohydroxylated products, while others tended to yield dehydroxylated and ketol metabolites.