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Oxygen limitation (hypoxia), arising as a key stress factor due to flooding, negatively affects plant development. Consequently, maintaining root growth under such stress is crucial for plant survival, yet we know little about the root system's adaptions to low‐oxygen conditions and its regulation by phytohormones. In this study, we examine the impact of hypoxia and, herein, the regulatory role of group VII ETHYLENE‐RESPONSE FACTOR (ERFVII) transcription factors on root growth in Arabidopsis. We found lateral root (LR) elongation to be actively maintained by hypoxia via ERFVII factors, as erfVII seedlings possess hypersensitivity towards hypoxia regarding their LR growth. Pharmacological inhibition of abscisic acid (ABA) biosynthesis revealed ERFVII‐driven counteraction of hypoxia‐induced inhibition of LR formation in an ABA‐dependent manner. However, postemergence LR growth under hypoxia mediated by ERFVIIs was independent of ABA. In roots, ERFVIIs mediate, among others, the induction of ABA‐degrading ABA 8′‐hydroxylases CYP707A1 expression. RAP2.12 could activate the pCYC707A1:LUC reporter gene, indicating, combined with single mutant analyses, that this transcription factor regulates ABA levels through corresponding transcript upregulation. Collectively, hypoxia‐induced adaptation of the Arabidopsis root system is shaped by developmental reprogramming, whereby ERFVII‐dependent promotion of LR emergence, but not elongation, is partly executed through regulation of ABA degradation. Summary statement: ERFVII pathway actively maintains abscisic acid homoeostasis, enabling a partial control of lateral root development under hypoxic conditions. [ABSTRACT FROM AUTHOR]

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm−2 was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low kLa of 0.076 h−1. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 103 cells cm−2, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 103 cells cm−2, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications. [ABSTRACT FROM AUTHOR]

Metabolism underpins all life‐sustaining processes and varies profoundly with body size, temperature and locomotor activity. A current theory explains some of the size‐dependence of metabolic rate (its mass exponent, b) through changes in metabolic level (L). We propose two predictive advances that: (a) combine the above theory with the evolved avoidance of oxygen limitation in water‐breathers experiencing warming, and (b) quantify the overall magnitude of combined temperatures and degrees of locomotion on metabolic scaling across air‐ and water‐breathers. We use intraspecific metabolic scaling responses to temperature (523 regressions) and activity (281 regressions) in diverse ectothermic vertebrates (fish, reptiles and amphibians) to show that b decreases with temperature‐increased L in water‐breathers, supporting surface area‐related avoidance of oxygen limitation, whereas b increases with activity‐increased L in air‐breathers, following volume‐related influences. This new theoretical integration quantitatively incorporates different influences (warming, locomotion) and respiration modes (aquatic, terrestrial) on animal energetics. [ABSTRACT FROM AUTHOR]

Virtually all aspects of the biology of ectotherms are size‐and temperature‐dependent. Aerobic metabolism is often proposed to explain such relationships, with oxygen limitation setting limits to heat tolerance and constraining growth. However, experimental tests of the role of oxygen in heat tolerance have yielded mixed results, suggesting that oxygen limitation may be important only in certain contexts and on certain time scales but not others.Here, using thermal death time curves, which incorporate the intensity and duration of heat stress, we quantify the ability to survive heat stress in multiple inbred lines of Drosophila melanogaster, under normal and low oxygen conditions. The lines were selected to differ markedly in body size and cell size, as these traits have been hypothesised to shape thermal tolerance via their effects on oxygen supply and demand.Low oxygen condition markedly reduced survival time across inbred lines, especially when flies were exposed to prolonged, mild heat stress.Variations in heat tolerance among lines were partly related to cell size and body size differences, especially under chronic exposure to high temperatures, under hypoxia and in flies that exhibit larger cell size, supporting the idea that differences in cell size affect the oxygen supply and demand via differences in surface area to volume ratio.Because differences in heat tolerance were manifested at different timescales, our results underscore the need to close the gap between responses to acute timescales, typically employed in laboratory studies, and chronic timescales, which are ecologically more relevant. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm−2 was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low kLa of 0.076 h−1. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 103 cells cm−2, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 103 cells cm−2, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications.

Forecasting long‐term consequences of global warming requires knowledge on thermal mortality and how heat stress interacts with other environmental stressors on different timescales. Here, we describe a flexible analytical framework to forecast mortality risks by combining laboratory measurements on tolerance and field temperature records. Our framework incorporates physiological acclimation effects, temporal scale differences and the ecological reality of fluctuations in temperature, and other factors such as oxygen. As a proof of concept, we investigated the heat tolerance of amphipods Dikerogammarus villosus and Echinogammarus trichiatus in the river Waal, the Netherlands. These organisms were acclimated to different temperatures and oxygen levels. By integrating experimental data with high‐resolution field data, we derived the daily heat mortality probabilities for each species under different oxygen levels, considering current temperatures as well as 1 and 2°C warming scenarios. By expressing heat stress as a mortality probability rather than a upper critical temperature, these can be used to calculate cumulative annual mortality, allowing the scaling up from individuals to populations. Our findings indicate a substantial increase in annual mortality over the coming decades, driven by projected increases in summer temperatures. Thermal acclimation and adequate oxygenation improved heat tolerance and their effects were magnified on longer timescales. Consequently, acclimation effects appear to be more effective than previously recognized and crucial for persistence under current temperatures. However, even in the best‐case scenario, mortality of D. villosus is expected to approach 100% by 2100, while E. trichiatus appears to be less vulnerable with mortality increasing to 60%. Similarly, mortality risks vary spatially: In southern, warmer rivers, riverine animals will need to shift from the main channel toward the cooler head waters to avoid thermal mortality. Overall, this framework generates high‐resolution forecasts on how rising temperatures, in combination with other environmental stressors such as hypoxia, impact ecological communities. [ABSTRACT FROM AUTHOR]

Oxygen‐balanced mixotrophy (OBM) is a novel type of microalgal cultivation that improves autotrophic productivity while reducing aeration costs and achieving high biomass yields on substrate. The scale‐up of this process is not straightforward, as nonideal mixing in large photobioreactors might have unwanted effects in cell physiology. We simulated at lab scale dissolved oxygen and glucose fluctuations in a tubular photobioreactor operated under OBM where glucose is injected at the beginning of the tubular section. We ran repeated batch experiments with the strain Galdieria sulphuraria ACUF 064 under glucose pulse feeding of different lengths, representing different retention times: 112, 71, and 21 min. During the long and medium tube retention time simulations, dissolved oxygen was depleted 15–25 min after every glucose pulse. These periods of oxygen limitation resulted in the accumulation of coproporphyrin III in the supernatant, an indication of disruption in the chlorophyll synthesis pathway. Accordingly, the absorption cross‐section of the cultures decreased steeply, going from values of 150–180 m2 kg−1 at the end of the first batch down to 50–70 m2 kg−1 in the last batches of both conditions. In the short tube retention time simulation, dissolved oxygen always stayed above 10% air saturation and no pigment reduction nor coproporphyrin III accumulation were observed. Concerning glucose utilization efficiency, glucose pulse feeding caused a reduction of biomass yield on substrate in the range of 4%–22% compared to the maximum levels previously obtained with continuous glucose feeding (0.9 C‐g C‐g−1). The missing carbon was excreted to the supernatant as extracellular polymeric substances constituted by carbohydrates and proteins. Overall, the results point out the importance of studying large‐scale conditions in a controlled environment and the need for a highly controlled glucose feeding strategy in the scale‐up of mixotrophic cultivation. [ABSTRACT FROM AUTHOR]

A warming climate poses a fundamental problem for embryos that develop within eggs because their demand for oxygen (O2) increases much more rapidly with temperature than their capacity for supply, which is constrained by diffusion across the egg surface. Thus, as temperatures rise, eggs may experience O2 limitation due to an imbalance between O2 supply and demand. Here, we formulate a mathematical model of O2 limitation and experimentally test whether this mechanism underlies the upper thermal tolerance in large aquatic eggs. Using Chinook salmon (Oncorhynchus tshawytscha) as a model system, we show that the thermal tolerance of eggs varies systematically with features of the organism and environment. Importantly, this variation can be precisely predicted by the degree to which these features shift the balance between O2 supply and demand. Equipped with this mechanistic understanding, we predict and experimentally confirm that the thermal tolerance of these embryos in their natural habitat is substantially lower than expected from laboratory experiments performed under normoxia. More broadly, our biophysical model of O2 limitation provides a mechanistic explanation for the elevated thermal sensitivity of fish embryos relative to other life stages, global patterns in egg size and the extreme fecundity of large teleosts.

Female-biased mortality has been consistently reported in Pacific salmon during their adult upriver migration. We collected coho salmon (Oncorhynchus kisutch (Walbaum, 1792)) upon arrival at their spawning grounds to test whether females are more prone to cardiac oxygen limitations following exercise stress. We used a surgical approach to periodically sample arterial and venous blood over 48 h following recovery from a chase protocol to induce maximum metabolic rate. We found no significant differences in arterial or venous partial pressures of O2 between males and females. Female salmon had significantly elevated plasma cortisol levels but there were no effects of sex on either plasma lactate or K+. Our data show that female coho salmon do not suffer oxygen limitations to the spongy myocardium after a single exercise event at moderate temperatures (14 °C)—at least not when arriving to their spawning grounds. This study found no clear support for a cardiac oxygen limitation underlying elevated female mortality in Pacific salmon. Neither, however, does our study design nor specific findings allow us to rule out cardiac limitations in these fish. Future work should address whether potential oxygen limitations to the spongy myocardium at high temperatures or oxygen limitations to the compact myocardium via coronary blood flow contribute to female-biased mortality earlier on the migratory route. [ABSTRACT FROM AUTHOR]

Background: Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. Results: Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L−1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. Conclusions: The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production. [ABSTRACT FROM AUTHOR]

In large‐scale bioreactors, there is often insufficient mixing and as a consequence, cells experience uneven substrate and oxygen levels that influence product formation. In this study, the influence of dissolved oxygen (DO) gradients on the primary and secondary metabolism of a high producing industrial strain of Penicillium chrysogenum was investigated. Within a wide range of DO concentrations, obtained under chemostat conditions, we observed different responses from P. chrysogenum: (i) no influence on growth or penicillin production (>0.025 mmol L−1); (ii) reduced penicillin production, but no growth limitation (0.013–0.025 mmol L−1); and (iii) growth and penicillin production limitations (<0.013 mmol L−1). In addition, scale down experiments were performed by oscillating the DO concentration in the bioreactor. We found that during DO oscillation, the penicillin production rate decreased below the value observed when a constant DO equal to the average oscillating DO value was used. To understand and predict the influence of oxygen levels on primary metabolism and penicillin production, we developed a black box model that was linked to a detailed kinetic model of the penicillin pathway. The model simulations represented the experimental data during the step experiments; however, during the oscillation experiments the predictions deviated, indicating the involvement of the central metabolism in penicillin production. [ABSTRACT FROM AUTHOR]

Rhizobium sp. IRBG74 is a nitrogen-fixing symbiont of Sesbania cannabina and a growth-promoting endophyte of rice, thus making it a good model to compare rhizobial interactions with legumes and cereals. In this report, we show that Rhizobium sp. IRBG74 forms biofilms on the roots of S. cannabina and rice. A mutant defective in biofilm formation was identified by screening a transposon mutant library. The transposon insertion was in thiQ , part of the thiBPQ operon that encodes the components of a thiamine/thiamine pyrophosphate ABC transporter. Complementation with thiBPQ partially restored biofilm formation. Addition of thiamine in growth media led to repression of thiC expression in the wild-type strain but not in the thiQ mutant. These results suggest that thiBPQ is involved in thiamine/TPP transport in Rhizobium sp. IRBG74. Using a GUS reporter, we show that the expression of thiC is significantly higher in biofilm as compared to cells in planktonic growth. Based on these results, we propose that Rhizobium sp. IRBG74 is thiamine-limited and requires thiamine transport for efficient biofilm formation and plant colonization. Thiamine synthesis in aerobic bacteria such as Rhizobium requires O2 and thus could be inhibited in the microaerobic/anaerobic conditions in biofilms. [ABSTRACT FROM AUTHOR]

Industrial-sized fermenters differ from the laboratory environment in which bioprocess development initially took place. One of the issues that can lead to reduced productivity on a large scale or even early termination of the process is the presence of bioreactor heterogeneities. This work proposes and adopts a design–build–test–learn-type workflow that estimates the substrate, oxygen, and resulting growth heterogeneities through a compartmental modelling approach and maps Yarrowia lipolytica-specific behavior in this relevant range of conditions. The results indicate that at a growth rate of 0.1 h−1, the largest simulated volume (90 m3) reached partial oxygen limitation. Throughout the fed-batch, the cells experienced dissolved oxygen values from 0 to 75% and grew at rates of 0 to 0.2 h−1. These simulated large-scale conditions were tested in small-scale cultivations, which elucidated a transcriptome with a strong downregulation of various transporter and central carbon metabolism genes during oxygen limitation. The relation between oxygen availability and differential gene expression was dynamic and did not show a simple on–off behavior. This indicates that Y. lipolytica can differentiate between different available oxygen concentrations and adjust its transcription accordingly. The workflow presented can be used for Y. lipolytica-based strain engineering, thereby accelerating bioprocess development. [ABSTRACT FROM AUTHOR]

Spatial heterogeneity is a pervasive feature of soils, affecting the distribution of carbon sources as well as their microbial consumers. Heterogeneous addition of substrates typically results in delayed microbial growth compared to homogeneous addition, and this effect has frequently been attributed to spatial separation of microorganisms from their food. We investigated the importance of two other potential causes of this effect, the availability of nutrients and oxygen, by measuring heat and CO 2 release along with O 2 consumption from soil samples after homogeneous or heterogeneous addition of glucose as well as with or without further addition of a nutrient solution. We then employed a microbial-explicit model to quantitatively interpret our observations. The results revealed that delayed growth after spatially heterogeneous substrate addition was primarily caused by nutrient limitation. While sufficient co-location of all entities - substrate, microorganisms, and nutrients - is required for optimal growth, spatial separation of glucose and microorganisms only played a minor role in our experiment. Model simulations captured the dynamics based on aerobic growth and maintenance, utilizing a simple formulation of nutrient limitation coupled with dynamic transition of microbes between activity and dormancy. The model predicted an overall lower microbial activity over the course of the incubation in treatments with heterogeneous substrate addition. Despite reduced rates, neither the experimental carbon and energy balances nor modeling showed an effect of heterogeneity on the growth efficiency after 50 h of incubation. In all treatments, energy use efficiency exceeded carbon use efficiency by 9–21%. We found no evidence of anaerobiosis. The application of a bioenergetic framework facilitated the interpretation of complex experimental data and quantitatively captured the mechanisms underlying the effects of spatial heterogeneity. • Heterogeneous glucose addition induced lower activity than homogeneous addition. • This effect of spatial substrate heterogeneity was caused by nutrient limitation. • The ratio of heat to CO 2 release was consistent with efficient aerobic growth. • EUE exceeded CUE and both were unaffected by spatial substrate heterogeneity. • A dynamic microbial-explicit model quantitatively captured the observed dynamics. [ABSTRACT FROM AUTHOR]

Abstract In large‐scale bioreactors, there is often insufficient mixing and as a consequence, cells experience uneven substrate and oxygen levels that influence product formation. In this study, the influence of dissolved oxygen (DO) gradients on the primary and secondary metabolism of a high producing industrial strain of Penicillium chrysogenum was investigated. Within a wide range of DO concentrations, obtained under chemostat conditions, we observed different responses from P. chrysogenum: (i) no influence on growth or penicillin production (>0.025 mmol L−1); (ii) reduced penicillin production, but no growth limitation (0.013–0.025 mmol L−1); and (iii) growth and penicillin production limitations (

In insects and other ectotherms, cold temperatures cause a coma resulting from loss of neuromuscular function, during which ionic and metabolic homeostasis are progressively lost. Cold adaptation improves homeostasis during cold exposure, but the ultimate targets of selection are still an open question. Cold acclimation and adaptation remodels mitochondrial metabolism in insects, suggesting that aerobic energy production during cold exposure could be a target of selection. Here, we test the hypothesis that cold adaptation improves the ability to maintain rates of aerobic energy production during cold exposure by using 31 P NMR on live flies. Using lines of Drosophila melanogaster artificially selected for fast and slow recovery from a cold coma, we show that cold exposure does not lower ATP levels and that cold adaptation does not alter aerobic ATP production during cold exposure. Cold-hardy and cold-susceptible lines both experienced a brief transition to anaerobic metabolism during cooling, but this was rapidly reversed during cold exposure, suggesting that oxidative phosphorylation was sufficient to meet energy demands below the critical thermal minimum, even in cold-susceptible flies. We thus reject the hypothesis that performance under mild low temperatures is set by aerobic ATP supply limitations in D. melanogaster, excluding oxygen and capacity limitation as a weak link in energy supply. This work suggests that the modulations to mitochondrial metabolism resulting from cold acclimation or adaptation may arise from selection on a biosynthetic product(s) of those pathways rather than selection on ATP supply during cold exposure.

The previously studied geographic variability in the growth patterns of different European sardine or European pilchard (Sardina pilchardus) populations has been attributed to the trophic status and productivity of the various ecosystems, as well as the genetic distance among populations. However, in the face of ocean warming and its multifaceted effects on marine populations and fisheries, it is interesting to explore growth patterns through the prism of sea temperature and dissolved oxygen. Here, data on the asymptotic length, growth coefficient, and maximum reported age of 47 Atlantic and Mediterranean sardine populations, covering the entire geographical range of its distribution, were extracted from published sources and were correlated with regional sea surface temperature and dissolved oxygen. Asymptotic length was negatively related to sea temperature and more strongly positively related to oxygen levels, indicating that sardines grow to larger body size in cooler waters that are more oxygenated. Within the context of climate change, the link of intraspecific growth variability with temperature and oxygen draws attention to the adverse effects this might have on many fish biological characteristics in a warming future. [ABSTRACT FROM AUTHOR]

Cobalamin (VB 12 ) is in enormous demand across the fields of medicine, food, and feed additives. However, the oxygen supply plays a critical role in VB 12 biosynthesis by Ensifer adhaerens Casida A and has been identified as a bottleneck for economical substrate consumption. This study elucidates the relationship between oxygen limitation and VB 12 accumulation with transcriptomic and metabolomic analyses. Under oxygen limitation, E. adhaerens enhances oxygen transport and storage by increasing expression of flavin hemoglobin (Hmp), which was up-regulated 6-fold at 24 h of oxygen restriction compared to the oxygen restriction of 4 h (p < 0.01). Because of the cofactor of Hmp is heme, the demand for heme increases, leading to the upregulation of genes in the heme biosynthesis pathway. Similarly, genes involved in biosynthesis of its precursor, 5-ALA, were upregulated as well. 5-ALA is also a direct precursor of VB 12 , further leading to the upregulation of genes in the VB 12 biosynthesis pathway. This process initiates biosynthesis and accumulation of VB 12 . As VB 12 and heme biosynthesis progresses, genes associated with the biosynthesis and transportation pathways of compounds related to their biosynthesis were likewise upregulated, including genes involved in S-adenosyl methionine (SAM) biosynthesis, and the transport of Fe 2+ and Co 2+ . Additionally, amino acids and organic acids associated with biosynthesis were also extensively consumed, such as methionine, which is used for synthesizing SAM, decreased by 310% after 24 h of oxygen limitation compared to 20% dissolved oxygen (p < 0.05). At the same time, genes related to growth-associated metabolic pathways, such as pentose phosphate pathway (PPP), were significantly downregulated. Therefore, the potential mechanism by which E. adhaerens accumulates VB 12 under oxygen-limited conditions by enhancing Hmp expression, which facilitates the porphyrin metabolic pathway and promotes VB 12 biosynthesis. This research provides valuable insights for increasing VB 12 production through metabolic engineering and process optimization., (© 2024 Wiley‐VCH GmbH.)

Abstract Ethyl acetate is currently produced from fossil carbon resources. This ester could also be microbially synthesized from sugar‐rich wastes of the food industry. Wild‐type strains with GRAS status are preferred for such applications. Production of ethyl acetate by wild‐type yeasts has been repeatedly reported, but comparative studies with several strains at various induction modes are largely missing. Here, synthesis of ethyl acetate by three yeasts with GRAS status, Kluyveromyces marxianus DSM 5422, Cyberlindnera jadinii DSM 2361 and Wickerhamomyces anomalus DSM 6766, was studied under identical and well‐defined conditions in an aerated bioreactor, by inducing the ester synthesis via iron or oxygen limitation. Balancing the ester synthesis was based on measured concentrations of ethyl acetate in the exhaust gas, delivering masses of synthesized ester and synthesis rates in a high temporal resolution. All tested yeasts synthesized ethyl acetate under these conditions, but the intensity varied with the strain and induction mode. The highest yields were achieved under iron limitation with K. marxianus (0.182 g g–1) and under oxygen limitation with W. anomalus (0.053 g g–1). Iron limitation proved to be the better inducer for ester synthesis while oxygen limitation favored ethanol formation. K. marxianus DSM 5422 was the most potent producer of ethyl acetate exhibiting the highest biomass‐specific synthesis rate of 0.5 g g–1h–1 under moderate iron limitation.

In the present study, the effect of employing the increasing- aeration strategy (IAS) in the oxygen-limited situation and proportionate to increasing oxygen demand of the fungus Schizophyllum commune (S. commune) has been investigated in both stirred tank (STB) and bubble column (BCB) bioreactors. The purpose was to enhance schizophyllan (SPG) production by preventing oxygen starvation, improve mixing conditions of pseudoplastic culture, and intensify shear stress on fungus pellets to release SPG. At first, a constant-aeration rate of 0.08 vvm was implemented in both bioreactors to evaluate the new strategy compared to the previously studied methods. In the second set of experiments with IAS, along with the increasing oxygen demand of culture, the inlet airflow was increased gradually, while the dissolved oxygen (DO) was maintained higher than zero and below 1%. Using IAS in STB significantly raised productivity by about 100% in 96 h from 0.035 to 0.073 g/L.h. Also, employing this strategy in BCB led to a 30% increase in the maximum SPG production from 3.2 to 4.2 g/L. IAS can effectively help handle the operation of S. commune cultivation on a large scale by improving mixing conditions, mass transfer, and shear stress in both bioreactor types. This method had a significant impact on STB cultivation and its productivity so that it can be a practical approach to SPG's industrial production. [ABSTRACT FROM AUTHOR]

Some microorganisms with the nitrification function can carry out nitrogen conversion under oxygen-limited conditions. In this study, a continuous-flow biofilm reactor was operated to investigate the removal of ammonium nitrogen (NH4-N) under microaerobic or anoxic conditions. After more than 100 days of long-term operation, the NH4-N removal rate and removal percentage reached 2.92 mg/(L·h) and 72.3%, respectively. In the batch experiments, NH4-N removal was achieved regardless of the presence or absence of nitrite. The main microorganisms relating to nitrogen metabolism in the system were Nitrospira, Nitrosomonas, Candidatus Kuenenia, and denitrifying bacteria. The metagenomics analysis indicated that these functional microorganisms constituted the main nitrogen metabolic network in the system, and microorganisms such as Rhodanobacter, Nitrosomonas, Defluvii, and Cyanobacteria had the possible capacity for oxygen production. Without organic carbon in the synthetic wastewater, heterotrophs including denitrifying bacteria might utilize organic carbon such as extracellular polymeric substances produced by autotrophs. The removal of NH4-N under oxygen-limited conditions might be achieved through interactions among nitrifying bacteria, anammox bacteria, and heterotrophs, which deserves further investigation. [ABSTRACT FROM AUTHOR]

Industrial-sized fermenters differ from the laboratory environment in which bioprocess development initially took place. One of the issues that can lead to reduced productivity on a large scale or even early termination of the process is the presence of bioreactor heterogeneities. This work proposes and adopts a design–build–test–learn-type workflow that estimates the substrate, oxygen, and resulting growth heterogeneities through a compartmental modelling approach and maps Yarrowia lipolytica-specific behavior in this relevant range of conditions. The results indicate that at a growth rate of 0.1 h−1, the largest simulated volume (90 m3) reached partial oxygen limitation. Throughout the fed-batch, the cells experienced dissolved oxygen values from 0 to 75% and grew at rates of 0 to 0.2 h−1. These simulated large-scale conditions were tested in small-scale cultivations, which elucidated a transcriptome with a strong downregulation of various transporter and central carbon metabolism genes during oxygen limitation. The relation between oxygen availability and differential gene expression was dynamic and did not show a simple on–off behavior. This indicates that Y. lipolytica can differentiate between different available oxygen concentrations and adjust its transcription accordingly. The workflow presented can be used for Y. lipolytica-based strain engineering, thereby accelerating bioprocess development.

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACD Cgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l -asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family. • C. glutamicum CR099 is resistant to high concentrations of pediocin PA-1. • Recombinant C. glutamicum CR099/pEKEx-pedACDCg produces of a compound with antimicrobial activity against Listeria sp. • The purified compound matches size and mass:charge ratio of commercial pediocin PA-1. • Low oxygen levels are favorable for production of active pediocin by C. glutamicum in batch fermentations. [ABSTRACT FROM AUTHOR]

Brain size varies dramatically, both within and across species, and this variation is often believed to be the result of trade‐offs between the cognitive benefits of having a large brain for a given body size and the energetic cost of sustaining neural tissue. One potential consequence of having a large brain is that organisms must also meet the associated high energetic demands. Thus, a key question is whether metabolic rate correlates with brain size. However, using metabolic rate to measure energetic demand yields a relatively instantaneous and dynamic measure of energy turnover, which is incompatible with the longer evolutionary timescale of changes in brain size within and across species. Morphological traits associated with oxygen consumption, specifically gill surface area, have been shown to be correlates of oxygen demand and energy use, and thus may serve as integrated correlates of these processes, allowing us to assess whether evolutionary changes in brain size correlate with changes in longer‐term oxygen demand and energy use. We tested how brain size relates to gill surface area in the blacktip shark Carcharhinus limbatus. First, we examined whether the allometric slope of brain mass (i.e., the rate that brain mass changes with body mass) is lower than the allometric slope of gill surface area across ontogeny. Second, we tested whether gill surface area explains variation in brain mass, after accounting for the effects of body mass on brain mass. We found that brain mass and gill surface area both had positive allometric slopes, with larger individuals having both larger brains and larger gill surface areas compared to smaller individuals. However, the allometric slope of brain mass was lower than the allometric slope of gill surface area, consistent with our prediction that the allometric slope of gill surface area could pose an upper limit to the allometric slope of brain mass. Finally, after accounting for body mass, individuals with larger brains tended to have larger gill surface areas. Together, our results provide clues as to how fishes may evolve and maintain large brains despite their high energetic cost, suggesting that C. limbatus individuals with a large gill surface area for their body mass may be able to support a higher energetic turnover, and, in turn, a larger brain for their body mass. [ABSTRACT FROM AUTHOR]

Oxygen limitation of aerobic metabolism is hypothesized to drive organismal thermal tolerance limits. Differences in oxygen availability in air and water may underlie observed differences in upper thermal tolerance of intertidal limpets if oxygen is limiting in submerged environments. We explored how cardiac performance (heart rate, breakpoint temperature [BPT], flat-line temperature [FLT], and temperature sensitivity) was affected by hyperoxia and hypoxia in the finger limpet, Lottia digitalis, under air exposure and submersion. Upper thermal tolerance limits were unchanged by increasing availability of oxygen, although air-exposed limpets were able to maintain cardiac function to higher temperatures than submerged limpets. Maximum heart rate did not increase with greater partial pressure of oxygen (Po2), suggesting that tissue Po2 levels are likely maximized during normoxia. Hypoxia reduced breakpoint BPTs and FLTs in air-exposed and submerged limpets and accentuated the difference in BPTs between the two groups through greater reductions in BPT in submerged limpets. Differences in respiratory structures and the degree to which thermal limits are already maximized may play significant roles in determining how oxygen availability influences upper temperature tolerance.

Abstract Background Recently, we showed that steady-state continuous cultures of S. stipitis follow the principles of growth on mixture of two complementary substrates. More precisely, when such cultures are fed with progressively higher concentrations of glucose s f at fixed dilution rate D = 0.1 h−1, oxygen mass-transfer coefficient k l a = 50 h−1, and oxygen solubility $$c_{\text{o}}^{*}$$ co∗ , they transition from glucose- to oxygen-limited growth through an intermediate dual-limited regime in which both glucose and oxygen are limiting, and ethanol is produced without loss of glucose. It is, therefore, of considerable interest to characterize the dual-limited regime. We found that the dual-limited regime occurs precisely when the operating parameters D, s f, k l a, and $$c_{\text{o}}^{*}$$ co∗ satisfy the relation $$Y_{\text{os}} < Ds_{\text{f}} /\left( {k_{\text{l}} a \cdot c_{\text{o}}^{*} } \right) < Y_{\text{os}}^{\prime }$$ Yos

Determining the life-history consequences for fishes living in extreme and variable environments will be vital in predicting the likely impacts of ongoing climate change on reef fish demography. Here, we compare size-at-age and maximum body size of two common reef fish species (Lutjanus ehrenbergii and Pomacanthus maculosus) between the environmentally extreme Arabian/Persian Gulf ('Arabian Gulf') and adjacent comparably benign Oman Sea. Additionally, we use otolith increment width profiles to investigate the influence of temperature, salinity and productivity on the individual growth rates. Individuals of both species showed smaller size-at-age and lower maximum size in the Arabian Gulf compared to conspecifics in the less extreme and less variable environment of the Oman Sea, suggesting a life-history trade-off between size and metabolic demands. Salinity was the best environmental predictor of interannual growth across species and regions, with low growth corresponding to more saline conditions. However, salinity had a weaker negative effect on interannual growth of fishes in the Arabian Gulf than in the Oman Sea, indicating Arabian Gulf populations may be better able to acclimate to changing environmental conditions. Temperature had a weak positive effect on the interannual growth of fishes in the Arabian Gulf, suggesting that these populations may still be living within their thermal windows. Our results highlight the potential importance of osmoregulatory cost in impacting growth, and the need to consider the effect of multiple stressors when investigating the consequences of future climate change on fish demography. [ABSTRACT FROM AUTHOR]

Ethyl acetate is currently produced from fossil carbon resources. This ester could also be microbially synthesized from sugar‐rich wastes of the food industry. Wild‐type strains with GRAS status are preferred for such applications. Production of ethyl acetate by wild‐type yeasts has been repeatedly reported, but comparative studies with several strains at various induction modes are largely missing. Here, synthesis of ethyl acetate by three yeasts with GRAS status, Kluyveromyces marxianus DSM 5422, Cyberlindnera jadinii DSM 2361 and Wickerhamomyces anomalus DSM 6766, was studied under identical and well‐defined conditions in an aerated bioreactor, by inducing the ester synthesis via iron or oxygen limitation. Balancing the ester synthesis was based on measured concentrations of ethyl acetate in the exhaust gas, delivering masses of synthesized ester and synthesis rates in a high temporal resolution. All tested yeasts synthesized ethyl acetate under these conditions, but the intensity varied with the strain and induction mode. The highest yields were achieved under iron limitation with K. marxianus (0.182 g g–1) and under oxygen limitation with W. anomalus (0.053 g g–1). Iron limitation proved to be the better inducer for ester synthesis while oxygen limitation favored ethanol formation. K. marxianus DSM 5422 was the most potent producer of ethyl acetate exhibiting the highest biomass‐specific synthesis rate of 0.5 g g–1h–1 under moderate iron limitation. [ABSTRACT FROM AUTHOR]

Biocatalysis using molecular oxygen as the electron acceptor has significant potential for selective oxidations at low cost. However, oxygen is poorly soluble in water, and its slow rate of mass transfer in the aqueous phase is a major obstacle, even for laboratory-scale syntheses. Oxygen transfer can be accelerated by vigorous mechanical methods, but these are often incompatible with biological catalysts. Gentler conditions can be achieved with shallow, high surface area bag reactors that are designed for single use and generally for specialized cell culture applications. As a less-expensive alternative to these high-end bioreactors, we describe repurposing inflatable shipping pillows with resealable valves to provide high surface area mixing under oxygen for preparative synthesis of glucosone (D-arabino-hexos-2-ulose) from D-glucose using non-growing Escherichia coli whole cells containing recombinant pyranose 2-oxidase (POX) as catalyst. Parallel reactions permitted systematic study of the effects of headspace composition (i.e., air vs 100% oxygen), cell density, exogenous catalase, and reaction volume in the oxidation of 10% glucose. Importantly, only a single charge of 100% oxygen is required for stoichiometric conversion on a multi-gram scale in 18 h with resting cells, and the conversion was successfully repeated with recycled cells. [ABSTRACT FROM AUTHOR]

Mycofactocin (MFT) is a recently discovered glycosylated redox cofactor, which has been associated with the detoxification of antibiotics in pathogenic mycobacteria, and, therefore, of potential medical interest. The MFT biosynthetic gene cluster is commonly found in mycobacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Since the MFT molecule is highly interesting for basic research and could even serve as a potential drug target, large-scale production of the molecule is highly desired. However, conventional shake flask cultivations failed to produce enough MFT for further biochemical characterization like kinetic studies and structure elucidation, and a more comprehensive study of cultivation parameters is urgently needed. Being a redox cofactor, it can be hypothesized that the oxygen transfer rate (OTR) is a critical parameter for MFT formation. Using the non-pathogenic strain Mycobacterium smegmatis mc2 155, shake flask experiments with online measurement of the oxygen uptake and the carbon dioxide formation, were conducted under different levels of oxygen supply. Using liquid chromatography and high-resolution mass spectrometry, a 4–8 times increase of MFT production was identified under oxygen-limited conditions, in both complex and mineral medium. Moreover, the level of oxygen supply modulates not only the overall MFT formation but also the length of the glycosidic chain. Finally, all results were scaled up into a 7 L stirred tank reactor to elucidate the kinetics of MFT formation. Ultimately, this study enables the production of high amounts of these redox cofactors, to perform further investigations into the role and importance of MFTs.

• We studied an acetoacetyl-CoA reductase with the highest NADH-preference so far described. • Residues between E37 and P41 are key for the NADH preference. • The expression of this enzyme enabled production of PHB as a fermentation product in Escherichia coli. Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (k cat NADH/K M NADH)/(k cat NADPH/K M NADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli , opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB. [ABSTRACT FROM AUTHOR]

The present study determined the effect of body mass and acclimation temperature (15–28°C) on oxygen consumption rate (ṀO2) and the size dependency of preferred temperature in European perch Perca fluviatilis. Standard metabolic rate (SMR) scaled allometrically with body mass by an exponent of 0.86, and temperature influenced SMR with a Q10 of 1.9 regardless of size. Maximum metabolic rate (MMR) and aerobic scope (MMR‐SMR) scaled allometrically with body mass by exponents of 0.75–0.88. The mass scaling exponents of MMR and aerobic scope changed with temperature and were lowest at the highest temperature. Consequently, the optimal temperature for aerobic scope decreased with increasing body mass. Notably, fish <40 g did not show a decrease aerobic scope with increasing temperature. Factorial aerobic scope (MMR × SMR−1) generally decreased with increasing temperatures, was unaffected by size at the lower temperatures, and scaled negatively with body mass at the highest temperature. Similar to the optimal temperature for aerobic scope, preferred temperature declined with increasing body mass, unaffectedly by acclimation temperature. The present study indicates a limitation in the capacity for oxygen uptake in larger fish at high temperatures. A constraint in oxygen uptake at high temperature may restrict the growth of larger fish with environmental warming, at least if food availability is not limited. Furthermore, behavioural thermoregulation may be contributing to regional changes in the size distribution of fish in the wild caused by global warming as larger individuals will prefer colder water at higher latitudes and at larger depths than smaller conspecifics with increasing environmental temperatures. [ABSTRACT FROM AUTHOR]

Juvenile growth trajectories evolve via the interplay of selective pressures on age and size at maturity, and developmental constraints. In insects, the moulting cycle is a major constraint on larval growth trajectories.Surface area to volume ratio of a larva decreases during growth, so renewal of certain surfaces by moulting is likely needed for the maintenance of physiological efficiency. A null hypothesis of isometry, implied by Dyar's Rule, would mean that the relative measures of growth remain constant across moults and instars.We studied ontogenetic changes and allometry in instar‐specific characteristics of larval growth in 30 lepidopteran species in a phylogenetic comparative framework.Relative instar‐specific mass increments (RMI) typically, but not invariably, decreased across instars. Ontogenetic change in RMIs varied among families with little within‐family variation. End‐of‐instar growth deceleration (GD) became stronger with increasing body size across instars. Across‐instar change in GD was conserved across taxa. Ontogenetic allometry was generally non‐isometric in both RMI and GD.Results indicate that detailed studies on multiple species are needed for generalizations concerning growth trajectory evolution. Developmental and physiological mechanisms affecting growth trajectory evolution show different degrees of evolutionary conservatism, which must be incorporated into models of age and size at maturation. [ABSTRACT FROM AUTHOR]

Ectotherms can become physiologically challenged when performing oxygen-demanding activities (e.g., flight) across differing environmental conditions, specifically temperature and oxygen levels. Achieving a balance between oxygen supply and demand can also depend on the cellular composition of organs, which either evolves or changes plastically in nature; however, this hypothesis has rarely been examined, especially in tracheated flying insects. The relatively large cell membrane area of small cells should increase the rates of oxygen and nutrient fluxes in cells; however, it does also increase the costs of cell membrane maintenance. To address the effects of cell size on flying insects, we measured the wing-beat frequency in two cell-size phenotypes of Drosophila melanogaster when flies were exposed to two temperatures (warm/hot) combined with two oxygen conditions (normoxia/hypoxia). The cell-size phenotypes were induced by rearing 15 isolines on either standard food (large cells) or rapamycin-enriched food (small cells). Rapamycin supplementation (downregulation of TOR activity) produced smaller flies with smaller wing epidermal cells. Flies generally flapped their wings at a slower rate in cooler (warm treatment) and less-oxygenated (hypoxia) conditions, but the small-cell-phenotype flies were less prone to oxygen limitation than the large-cell-phenotype flies and did not respond to the different oxygen conditions under the warm treatment. We suggest that ectotherms with small-cell life strategies can maintain physiologically demanding activities (e.g., flight) when challenged by oxygen-poor conditions, but this advantage may depend on the correspondence among body temperatures, acclimation temperatures and physiological thermal limits.

Abstract Trichoderma reesei is well known as an industrial workhorse fungus in cellulase production. The low dissolved oxygen supply in the highly viscous medium of T. reesei remains a major bottleneck that hampers growth and cellulase production in submerged fermentation. Vitreoscilla hemoglobin (VHb) has been demonstrated to improve metabolism and protein production in different heterologous hosts under hypoxic conditions, but the use of VHb in T. reesei remains uninvestigated. This study examines the effect of VHb in improving T. reesei performance in submerged fermentation. The VHb gene (vgb)-expressing cassette was successfully transformed into the TU-6 strain, integrated into the genome of T. reesei, and functionally expressed with biological activity, which was confirmed by carbon monoxide difference analysis. Compared to the parent strain, the expression of VHb increased the glucose consumption rate of the transformant. Moreover, in cellulase-inducing medium total protein secretion of the VHb expressing strain was 2.2-fold of the parental strain and the filter paper cellulase activity was increased by 58% under oxygen-limiting conditions. In summary, our results demonstrate that VHb has beneficial effects on improving total protein secretion and cellulase activity of T. reesei in submerged fermentation.

Abstract Background In the phase of oxygen limitation during Aspergillus niger fermentation, the cell growth decreased, while the yield of glucoamylase increased continuously and significantly. Explanation on the changes of transcriptome profile during this process may improve our understanding on mechanisms of cell adaption and enzyme production in A. niger. Results The transcriptomic data from 4 time points in oxygen limitation process of a glucoamylase production A. niger strain were analyzed. Hierarchical clustering of all samples showed that the strongest transcriptional response occurred between the early and middle stage of oxygen limitation. 515 differentially expressed genes (DEGs) were identified and were clustered into 12 expression patterns. Continuously down-regulated DEGs were significantly enriched in GO terms of the ribosome, translation, and aminoacyl-tRNA biosynthesis, and continuously up-regulated DEGs were mainly involved in GO terms of fatty acid catabolism, N-acetyltransferase activity, lipase activity, and carboxylesterase activity. Pyruvate kinase and asparagine synthetase which related to the biosynthesis of the main amino acid composition of the enzyme were significantly up-regulated. Sterol-regulatory element-binding proteins (SREBP) transcription factor SrbB was one of the most up-regulated proteins, indicating its important roles in hypoxia fermentation of A. niger. Conclusion Comparative transcriptome data analysis of the fermentation revealed that the overall reduced biosynthesis of translation machine and fatty acid, acceleration of fatty acid catabolism, and increased synthesis of main amino acid compositions of glucoamylase are the key transcriptional changes during the oxygen limitation fermentation process of A. niger.

Declining body size in fishes and other aquatic ectotherms associated with anthropogenic climate warming has significant implications for future fisheries yields, stock assessments and aquatic ecosystem stability. One proposed mechanism seeking to explain such body-size reductions, known as the gill oxygen limitation (GOL) hypothesis, has recently been used to model future impacts of climate warming on fisheries but has not been robustly empirically tested. We used brook trout (Salvelinus fontinalis), a fast-growing, cold-water salmonid species of broad economic, conservation and ecological value, to examine the GOL hypothesis in a long-term experiment quantifying effects of temperature on growth, resting metabolic rate (RMR), maximum metabolic rate (MMR) and gill surface area (GSA). Despite significantly reduced growth and body size at an elevated temperature, allometric slopes of GSA were not significantly different than 1.0 and were above those for RMR and MMR at both temperature treatments (15°C and 20°C), contrary to GOL expectations. We also found that the effect of temperature on RMR was time-dependent, contradicting the prediction that heightened temperatures increase metabolic rates and reinforcing the importance of longer-term exposures (e.g. >6 months) to fully understand the influence of acclimation on temperature-metabolic rate relationships. Our results indicate that although oxygen limitation may be important in some aspects of temperature-body size relationships and constraints on metabolic supply may contribute to reduced growth in some cases, it is unlikely that GOL is a universal mechanism explaining temperature-body size relationships in aquatic ectotherms. We suggest future research focus on alternative mechanisms underlying temperature-body size relationships, and that projections of climate change impacts on fisheries yields using models based on GOL assumptions be interpreted with caution., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Similar to humans, insects lose their physical and physiological capacities with age, which makes them a convenient study system for human ageing. Although insects have an efficient oxygen-transport system, we know little about how their flight capacity changes with age and environmental oxygen conditions. We measured two types of locomotor performance in ageing Drosophila melanogaster flies: the frequency of wing beats and the capacity to climb vertical surfaces. Flight performance was measured under normoxia and hypoxia. As anticipated, ageing flies showed systematic deterioration of climbing performance, and low oxygen impeded flight performance. Against predictions, flight performance did not deteriorate with age, and younger and older flies showed similar levels of tolerance to low oxygen during flight. We suggest that among different insect locomotory activities, flight performance deteriorates slowly with age, which is surprising, given that insect flight is one of the most energy-demanding activities in animals. Apparently, the superior capacity of insects to rapidly deliver oxygen to flight muscles remains little altered by ageing, but we showed that insects can become oxygen limited in habitats with a poor oxygen supply (e.g., those at high elevations) during highly oxygen-demanding activities such as flight.

A pUC‐derived replicon inducible by oxygen limitation was designed and tested in fed‐batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from Ptrc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (YpDNA/X) were performed and the initial hypothesis was probed. Furthermore, in high cell‐density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen‐responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and YpDNA/X efficiently increased after the shift to the microaerobic regime in fed‐batch cultures in a 1 L bioreactor. The YpDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed‐batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale‐up of plasmid DNA production processes in which mass transfer limitations will not represent an issue. [ABSTRACT FROM AUTHOR]

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA−, which, together with W3110recA−vgb+, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production. [ABSTRACT FROM AUTHOR]

Global warming appears to favour smaller-bodied organisms, but whether larger species are also more vulnerable to thermal extremes, as suggested for past mass-extinction events, is still an open question. Here, we tested whether interspecific differences in thermal tolerance (heat and cold) of ectotherm organisms are linked to differences in their body mass and genome size (as a proxy for cell size). Since the vulnerability of larger, aquatic taxa to warming has been attributed to the oxygen limitation hypothesis, we also assessed how body mass and genome size modulate thermal tolerance in species with contrasting breathing modes, habitats and life stages. A database with the upper (CTmax) and lower (CTmin) critical thermal limits and their methodological aspects was assembled comprising more than 500 species of ectotherms. Our results demonstrate that thermal tolerance in ectotherms is dependent on body mass and genome size and these relationships became especially evident in prolonged experimental trials where energy efficiency gains importance. During long-term trials, CTmax was impaired in larger-bodied water-breathers, consistent with a role for oxygen limitation. Variation in CTmin was mostly explained by the combined effects of body mass and genome size and it was enhanced in larger-celled, air-breathing species during long-term trials, consistent with a role for depolarization of cell membranes. Our results also highlight the importance of accounting for phylogeny and exposure duration. Especially when considering long-term trials, the observed effects on thermal limits are more in line with the warming-induced reduction in body mass observed during long-term rearing experiments. This article is part of the theme issue 'Physiological diversity, biodiversity patterns and global climate change: testing key hypotheses involving temperature and oxygen'. [ABSTRACT FROM AUTHOR]

Ghost moths inhabiting the alpine meadows of the Tibetan Plateau are cold‐adapted stenothermal organisms that are susceptible to heat (dead within 7 days at 27 °C exposure). Exploring the metabolic basis of their heat susceptibility would extend our understanding of the thermal biology of alpine‐dwelling invertebrates. Here, gas chromatography–mass spectrometry‐based metabolomics was combined with physiological and transcriptional approaches to determine the metabolic mechanisms of heat susceptibility in Thitarodes xiaojinensis larvae. The metabolomics results showed that 27 °C heat stress impaired the Krebs cycle and lipolysis in T. xiaojinensis larvae, as demonstrated by the accumulation of intermediary metabolites. In addition, carbohydrate reserves were highly and exclusively consumed, and an anaerobic product, lactate, accumulated. This evidence suggested a strong reliance on glycolysis to anaerobically generate energy. The respiration rate and enzymatic activity test results indicated a deficiency in O2 metabolism; in addition, the Krebs cycle capacity was not decreased, and the metabolic flux through aerobic pathways was limited. These findings were further supported by the occurrence of hypoxia symptoms in midgut mitochondria (vacuolation and swelling) and increased transcription of hypoxia‐induced factor 1‐α. Overall, heat stress caused O2 limitation and depressed the overall intensity of aerobic metabolism in ghost moths, and less efficient anaerobic glycolysis was activated to sustain their energy supply. As carbohydrates were depleted, the energy supply became deficient. Our study presents a comprehensive metabolic explanation for the heat susceptibility of ghost moths and reveals the relationship between O2 metabolism and heat susceptibility in these larvae. [ABSTRACT FROM AUTHOR]

• Sodium acetate is the best buffer to keep pH above the critical value in resting cells operation. • A specific OUR of 6.3·10−6 mol·g−1·s−1 prevents OTR from being the rate limiting step. • Under non-limited oxygen conditions, the highest initial specific DHA production rate is 4.8 g DHA ·g X −1 h−1. The production of dihydroxyacetone (DHA) from glycerol using Gluconobacter oxydans in resting cells is studied. An appropriate buffer has been selected to avoid the negative impact of pH decline on glycerol conversion caused by glyceric acid by-product accumulation in broth. Besides, the simultaneous influence of biomass concentration and oxygen transport rate has been studied in resting cells, determining the maximum specific oxygen uptake rate that ensures the highest culture activity. This parameter lets us know whether oxygen transport rate is the DHA production rate limiting factor. Under this limitation, efforts to increase DHA production rate by increasing biomass concentration are in vain. Surprisingly, glyceric acid production is not affected by oxygen transport rate limitation. Therefore, oxygen transport rate must be adjusted to biomass concentration to successfully obtain an increase in DHA production rate. [ABSTRACT FROM AUTHOR]

Mainstream partial nitritation was studied at 10 °C in a moving bed biofilm reactor treating synthetic wastewater containing both nitrogen (≈40 mg L−1) and organic carbon at COD/N ratio ranging from 1.3 to 2.2. Three different control strategies were investigated to achieve partial nitritation. Initially, biofilm age was controlled by incorporating a media replacement strategy. Next, separately from the media replacement, oxygen limited conditions were investigated and finally pH control was incorporated together with oxygen limitation. Successful partial nitritation was achieved only by combining oxygen limitation with pH control. The average NH 4 -N concentration was equal to 16.0 ± 1.6 mg L−1 and average NO 2 -N concentration was equal to 15.7 ± 2.4 mg L−1 during steady state partial nitritation. The average residual NO 3 -N concentration was equal to 2.6 ± 2.2 mg L−1. The results obtained from this study prove for the first time that partial nitritation can be successfully controlled in a biofilm reactor treating wastewater with low nitrogen concentration, relatively high COD/N ratio and at low temperature. An algorithm for dynamic process control of partial nitritation has been also developed. Image 1 • Partial nitritation was achieved at 10 °C in MBBR reactor. • Average NH 4 -N, NO 2 -N concentrations equaled to 16.0 ± 1.6 mg L−1and 15.7 ± 2.4 mg L−1. • DO/TAN of 0.06 maintained NH 4 -N oxidation at 57 ± 5% but did not produce nitrite. • FA concentration of 1.1 ± 0.2 mg NH 3 -N L−1 inhibited NOB activity. • Novel partial nitritation process control algorithm was developed. [ABSTRACT FROM AUTHOR]

Abstract Overflow metabolism is a common phenomenon observed at higher glycolytic flux in many bacteria, yeast (known as Crabtree effect), and mammalian cells including cancer cells (known as Warburg effect). This phenomenon has recently been characterized as the trade-offs between protein costs and enzyme efficiencies based on coarse-graining approaches. Moreover, it has been recognized that the glycolytic flux increases as the source of energy generation changes from energetically efficient respiration to inefficient respiro-fermentative or fermentative metabolism causing overflow metabolism. It is highly desired to clarify the metabolic regulation mechanisms behind such phenomena. Metabolic fluxes are located on top of the hierarchical regulation systems, and represent the outcome of the integrated response of all levels of cellular regulation systems. In the present article, we discuss about the different levels of regulation systems for the modulation of fluxes depending on the growth rate, growth condition such as oxygen limitation that alters the metabolism towards fermentation, and genetic perturbation affecting the source of energy generation from respiration to respiro-fermentative metabolism in relation to overflow metabolism. The intracellular metabolite of the upper glycolysis such as fructose 1,6-bisphosphate (FBP) plays an important role not only for flux sensing, but also for the regulation of the respiratory activity either directly or indirectly (via transcription factors) at higher growth rate. The glycolytic flux regulation is backed up (enhanced) by unphosphorylated EIIA and HPr of the phosphotransferase system (PTS) components, together with the sugar-phosphate stress regulation, where the transcriptional regulation is further modulated by post-transcriptional regulation via the degradation of mRNA (stability of mRNA) in Escherichia coli. Moreover, the channeling may also play some role in modulating the glycolytic cascade reactions. Highlights • Flux sensing metabolite such as FBP influences the respiratory activity • Source of energy generation affects the glycolytic flux and overflow metabolism • Sugar-phosphate stress, gene expression, and mRNA stability determine the flux [ABSTRACT FROM AUTHOR]

The present study describes the microbial production of polyhydroxyalkanoates (PHA) from thermally pre-treated sewage sludge at pilot scale level, investigating for the first time the effect of the organic loading rate (OLR) under oxygen limitation on biomass storage properties and kinetics. Polymer characteristics have been also evaluated. The selection/enrichment of PHA-storing biomass was successfully achieved in a Sequencing Batch Reactor (SBR) under short hydraulic retention time (HRT; 2 days). Low OLR (2.05 g COD/L d) was ideal for the selection of an efficient PHA-producing consortium cultivated under limited oxygen availability. In the fed-batch accumulation conducted under high DO regime, such biomass was characterized by 51% of PHA content on cell dry weight, with a related storage yield (Y P/S batch) of 0.61 COD PHA /COD S. On the contrary, medium OLR (4.56 g COD/L d) was not technically feasible to sustain the required consortium's selection under low DO regime. The PHA produced by biomass cultivated under low DO regime was characterized higher thermal stability and crystalline domain compared to PHA traditionally produced under high DO regime. The mass balance assessment highlighted a global yield of 51 g PHA/kg VS (volatile solids of thickened sludge), which was 9% lower than yield obtained under high DO regime, in the face of a realistic reduction of the energy cost of the process. [Display omitted] • Low DO regime sustained the PHA-storing biomass selection in SBR at low OLR. • High OLR was not technically suitable to select PHA-storing biomass at low DO. • Butyrate and valerate were preferentially consumed in the fed-batch accumulations. • The extracted PHA had high thermal stability and crystalline domain. • An overall PHA yield of 51 g PHA/g VS from sewage sludge was quantified. [ABSTRACT FROM AUTHOR]