Search Results (11)

Biocement can be achieved through the microbially induced carbonate precipitation (MICP) process. Such a method can potentially be utilized as an eco-friendly method for civil and environmental engineering applications such as soil ground improvement and wind erosion control of surface soil. In this method, one key step is the effective production of ureolytic bacteria. In previous laboratory and field studies, the cultivation and production of the bacteria used for the MICP were usually expensive and time-consuming. The purpose of this study was to optimize the cultivation method of the ureolytic bacteria (Sporosarcina pasteurii), and soil stabilization tests were conducted to verify the effectiveness of the cultured bacteria used to strengthen soil against the wind-induced erosion. Bacterial cultivation methods were studied by investigating the effects of different cultivation media and conditions. Testing variables included the types and concentrations of nitrogen sources (urea or NH₄Cl), pH values (7.5–9.5), cultivation conditions (batch or chemostat condition), and different carbon sources. It was found that, with the same amount of nitrogen source, the test with pure urea had the highest biomass yield, urease activity, and specific urease activity than the other tests with pure NH₄Cl or both NH₄Cl and urea. The use of urea as the nitrogen source in the media also led to an increase in pH, which was not found in the test with pure NH₄Cl. As for the factor of urea concentration, the tests with a higher urea concentration had a higher biomass yield, urease activity, and pH. The factor of pH values also played an important role. The test with an 8.5 initial pH value had a higher biomass yield, urease activity, and specific urease activity than the tests with 7.5 and 9.5 initial pH values. In the chemostat condition, the ureolytic bacteria could be effectively produced with urease activity up to 7 mmol/L/min, as compared with around 12 mmol/L/min activity in the batch condition. Thus, the optimum nitrogen source, pH value, and cultivation condition for the cultivation of Sporosarcina pasteurii was urea, 8.5, and batch condition, respectively. In addition, when soybean milk powder or milk powder was used as the carbon source, the urease activity was around 2.5 mmol/L/min, which is also high enough to be used for biocement. Full article

Magnetotactic bacteria (MTB) produce magnetosomes, which are membrane-embedded magnetic nanoparticles. Despite their technological applicability, the production of magnetite magnetosomes depends on the cultivation of MTB, which results in low yields. Thus, strategies for the large-scale cultivation of MTB need to be improved. Here, we describe a new approach for bioreactor cultivation of Magnetovibrio blakemorei strain MV-1^T. Firstly, a fed-batch with a supplementation of iron source and N₂O injection in 24-h pulses was established. After 120 h of cultivation, the production of magnetite reached 24.5 mg∙L⁻¹. The maximum productivity (16.8 mg∙L⁻¹∙day⁻¹) was reached between 48 and 72 h. However, the productivity and mean number of magnetosomes per cell decreased after 72 h. Therefore, continuous culture in the chemostat was established. In the continuous process, magnetite production and productivity were 27.1 mg∙L⁻¹ and 22.7 mg∙L⁻¹∙day⁻¹, respectively, at 120 h. This new approach prevented a decrease in magnetite production in comparison to the fed-batch strategy. Full article

Recirculation of solid digestate through digesters has been demonstrated to be a potential simple strategy to increase continuous stirred-tank reactor biogas plant efficiency. This study extended this earlier work and investigated solid digestate post-treatment using liquid isolated ligninolytic aerobic consortia in order to increase methane recovery during the recirculation. Based on sampling in several natural environments, an enrichment and selection method was implemented using a Lab-scale Automated and Multiplexed (an)Aerobic Chemostat system to generate ligninolytic aerobic consortia. Then, obtained consortia were further cultivated under liquid form in bottles. Chitinophagia bacteria and Sordariomycetes fungi were the two dominant classes of microorganisms enriched through these steps. Finally, these consortia where mixed with the solid digestate before a short-term aerobic post-treatment. However, consortia addition did not increase the efficiency of aerobic post-treatment of solid digestate and lower methane yields were obtained in comparison to the untreated control. The main reason identified is the respiration of easily degradable fractions (e.g., sugars, proteins, amorphous cellulose) by the selected consortia. Thus, this paper highlights the difficulties of constraining microbial consortia to sole ligninolytic activities on complex feedstock, such as solid digestate, that does not only contain lignocellulosic structures. Full article

Bioreactor scale-up from the laboratory scale to the industrial scale has always been a pivotal step in bioprocess development. However, the transition of a bioeconomy from innovation to commercialization is often hampered by performance loss in titer, rate and yield. These are often ascribed to temporal variations of substrate and dissolved oxygen (for instance) in the environment, experienced by microorganisms at the industrial scale. Oscillations in dissolved oxygen (DO) concentration are not uncommon. Furthermore, these fluctuations can be exacerbated with poor mixing and mass transfer limitations, especially in fermentations with filamentous fungus as the microbial cell factory. In this work, the response of glucose-limited chemostat cultures of an industrial Penicillium chrysogenum strain to different dissolved oxygen levels was assessed under both DO shift-down (60% → 20%, 10% and 5%) and DO ramp-down (60% → 0% in 24 h) conditions. Collectively, the results revealed that the penicillin productivity decreased as the DO level dropped down below 20%, while the byproducts, e.g., 6-oxopiperidine-2-carboxylic acid (OPC) and 6-aminopenicillanic acid (6APA), accumulated. Following DO ramp-down, penicillin productivity under DO shift-up experiments returned to its maximum value in 60 h when the DO was reset to 60%. The result showed that a higher cytosolic redox status, indicated by NADH/NAD+, was observed in the presence of insufficient oxygen supply. Consistent with this, flux balance analysis indicated that the flux through the glyoxylate shunt was increased by a factor of 50 at a DO value of 5% compared to the reference control, favoring the maintenance of redox status. Interestingly, it was observed that, in comparison with the reference control, the penicillin productivity was reduced by 25% at a DO value of 5% under steady state conditions. Only a 14% reduction in penicillin productivity was observed as the DO level was ramped down to 0. Furthermore, intracellular levels of amino acids were less sensitive to DO levels at DO shift-down relative to DO ramp-down conditions; this difference could be caused by different timescales between turnover rates of amino acid pools (tens of seconds to minutes) and DO switches (hours to days at steady state and minutes to hours at ramp-down). In summary, this study showed that changes in oxygen availability can lead to rapid metabolite, flux and productivity responses, and dynamic DO perturbations could provide insight into understanding of metabolic responses in large-scale bioreactors. Full article

Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for ¹³C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This “metabolic flux probe” (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for ¹³C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics. Full article

The crenarchaeal model organism Sulfolobus acidocaldarius is typically cultivated in shake flasks. Although shake flasks represent the state-of-the-art for the cultivation of this microorganism, in these systems crucial process parameters, like pH or substrate availability, are only set initially, but cannot be controlled during the cultivation process. As a result, a thorough characterization of growth parameters under controlled conditions is still missing for S. acidocaldarius. In this study, we conducted chemostat cultivations at 75 °C using a growth medium containing L-glutamate and D-glucose as main carbon sources. Different pH values and dilution rates were applied with the goal to physiologically characterize the organism in a controlled bioreactor environment. Under these controlled conditions a pH optimum of 3.0 was determined. Washout of the cells occurred at a dilution rate of 0.097 h⁻¹ and the optimal productivity of biomass was observed at a dilution rate of 0.062 h⁻¹. While both carbon sources were taken up by S. acidocaldarius concomitantly, a 6.6-fold higher affinity for L-glutamate was shown. When exposed to suboptimal growth conditions, S. acidocaldarius reacted with a change in the respiratory behavior and an increased trehalose production rate in addition to a decreased growth rate. Full article

Due to insufficient mass transfer and mixing issues, cells in the industrial-scale bioreactor are often forced to experience glucose feast/famine cycles, mostly resulting in reduced commercial metrics (titer, yield and productivity). Trehalose cycling has been confirmed as a double-edged sword in the Penicillium chrysogenum strain, which facilitates the maintenance of a metabolically balanced state, but it consumes extra amounts of the ATP responsible for the repeated breakdown and formation of trehalose molecules in response to extracellular glucose perturbations. This loss of ATP would be in competition with the high ATP-demanding penicillin biosynthesis. In this work, the role of trehalose metabolism was further explored under industrially relevant conditions by cultivating a high-yielding Penicillium chrysogenum strain, and the derived trehalose-null strains in the glucose-limited chemostat system where the glucose feast/famine condition was imposed. This dynamic feast/famine regime with a block-wise feed/no feed regime (36 s on, 324 s off) allows one to generate repetitive cycles of moderate changes in glucose availability. The results obtained using quantitative metabolomics and stoichiometric analysis revealed that the intact trehalose metabolism is vitally important for maintaining penicillin production capacity in the Penicillium chrysogenum strain under both steady state and dynamic conditions. Additionally, cells lacking such a key metabolic regulator would become more sensitive to industrially relevant conditions, and are more able to sustain metabolic rearrangements, which manifests in the shrinkage of the central metabolite pool size and the formation of ATP-consuming futile cycles. Full article

Lactic acid is the monomeric unit of polylactide (PLA), a bioplastic widely used in the packaging, automotive, food, and pharmaceutical industries. Previously, the yeast Komagataella phaffii was genetically modified for the production of lactate from glycerol. For this, the bovine L-lactate dehydrogenase- (LDH)-encoding gene was inserted and the gene encoding the pyruvate decarboxylase (PDC) was disrupted, resulting in the GLp strain. This showed a yield of 67% L-lactic acid and 20% arabitol as a by-product in batches with oxygen limitation. Following up on these results, the present work endeavored to perform a detailed study of the metabolism of this yeast, as well as perturbing arabitol synthesis in an attempt to increase lactic acid titers. The GLp strain was cultivated in a glycerol-limited chemostat at different dilution rates, confirming that the production of both lactic acid and arabitol is dependent on the specific growth rate (and consequently on the concentration of the limiting carbon source) as well as on the oxygen level. Moreover, disruption of the gene encoding arabitol dehydrogenase (ArDH) was carried out, resulting in an increase of 20% in lactic acid and a 50% reduction in arabitol. This study clarifies the underlying metabolic reasons for arabitol formation in K. phaffii and points to ways for improving production of lactic acid using K. phaffii as a biocatalyst. Full article

The blackwater stream of domestic wastewater contains energy and the majority of nutrients that can contribute to a circular economy. Hygienically safe and odor-free nutrient solution produced from anaerobically treated source-separated blackwater through an integrated post-treatment unit can be used as a source of liquid fertilizer. However, the high water content in the liquid fertilizer represents a storage or transportation challenge when utilized on agricultural areas, which are often situated far from the urban areas. Integration of microalgae into treated source-separated blackwater (BW) has been shown to effectively assimilate and recover phosphorus (P) and nitrogen (N) in the form of green biomass to be used as slow release biofertilizer and hence close the nutrient loop. With this objective, a lab-scale flat panel photobioreactor was used to cultivate Chlorella sorokiniana strain NIVA CHL 176 in a chemostat mode of operation. The growth of C. sorokiniana on treated source-separated blackwater as a substrate was monitored by measuring dry biomass concentration at a dilution rate of 1.38 d⁻¹, temperature of 37 °C and pH of 7. The results indicate that the N and P recovery rates of C. sorokiniana were 99 mg N L⁻¹d⁻¹ and 8 mg P L⁻¹d⁻¹ for 10% treated BW and reached 213 mg N L⁻¹d⁻¹ and 35 mg P L⁻¹d⁻¹, respectively when using 20% treated BW as a substrate. The corresponding biomass yield on light, N and P on the 20% treated BW substrate were 0.37 g (mol photon)⁻¹, 9.1 g g⁻¹ and 54.1 g g⁻¹, respectively, and up to 99% of N and P were removed from the blackwater. Full article

Together with other so-called “bio-plastics”, Polyhydroxyalkanoates (PHAs) are expected to soon replace established polymers on the plastic market. As a prerequisite, optimized process design is needed to make PHAs attractive in terms of costs and quality. Nowadays, large-scale PHA production relies on discontinuous fed-batch cultivation in huge bioreactors. Such processes presuppose numerous shortcomings such as nonproductive time for reactor revamping, irregular product quality, limited possibility for supply of certain carbon substrates, and, most of all, insufficient productivity. Therefore, single- and multistage continuous PHA biosynthesis is increasingly investigated for production of different types of microbial PHAs; this goes for rather crystalline, thermoplastic PHA homopolyesters as well as for highly flexible PHA copolyesters, and even blocky-structured PHAs consisting of alternating soft and hard segments. Apart from enhanced productivity and constant product quality, chemostat processes can be used to elucidate kinetics of cell growth and PHA formation under constant process conditions. Furthermore, continuous enrichment processes constitute a tool to isolate novel powerful PHA-producing microbial strains adapted to special environmental conditions. The article discusses challenges, potential and case studies for continuous PHA production, and shows up new strategies to further enhance such processes economically by developing unsterile open continuous processes combined with the application of inexpensive carbon feedstocks. Full article

The to-date studies on extreme halophiles were focused on shake flask cultivations. Bioreactor technology with quantitative approaches can offer a wide variety of biotechnological applications to exploit the special biochemical features of halophiles. Enabling industrial use of Haloferax mediterranei, finding the optima of cultivation parameters is of high interest. In general, process parameter optimizations were mainly carried out with laborious and time-consuming chemostat cultures. This work offers a faster alternative for process parameter optimization by applying temperature ramps and pH shifts on a halophilic continuous bioreactor culture. Although the hydraulic equilibrium in continuous culture is not reached along the ramps, the main effects on the activity from the dynamic studies can still be concluded. The results revealed that the optimal temperature range may be limited at the lower end by the activity of the primary metabolism pathways. At the higher end, the mass transfer of oxygen between the gaseous and the liquid phase can be limiting for microbial growth. pH was also shown to be a key parameter for avoiding overflow metabolism. The obtained experimental data were evaluated by clustering with multivariate data analyses. Showing the feasibility on a halophilic example, the presented dynamic methodology offers a tool for accelerating bioprocess development. Full article