The Sizes, Growth and Reproduction of Arrow Worms (Chaetognatha) in Light of the Gill-Oxygen Limitation Theory (GOLT)

The Chaetognatha are a marine invertebrate phylum including 132 extant, carnivorous species in nine families and two orders, but with unclear protostomian affinities in the animal kingdom. We document the gradual recognition of the distinctiveness of chaetognaths by early taxonomists, with some emphasis on the often-overlooked studies by Chinese marine biologists. The carnivorous arrow worms are understudied relative to their importance in the marine zooplankton, where they rank second in abundance after the herbivorous copepods. Although arrow worms lack gills or other dedicated respiratory organs, we show that the Gill-Oxygen Limitation Theory (GOLT) can be used to explain how temperature and respiration affect their growth and related life-history traits. Notably, we present a reappraisal of evidence for size–temperature relationships between and within chaetognath species, and for the relationship between their temperature-mediated oxygen demand and their growth patterns. Von Bertalanffy weight growth curves of Ferosagitta hispida (family: Sagittidae) based on earlier aquarium experiments by various authors are presented, which suggest (a) a good fit and (b) that the life span of chaetognaths is much lower than suggested by the authors of several published growth curves drawn onto length–frequency samples from the wild. In addition, we show that chaetognaths attain first maturity at a fraction of the maximum length they can attain that is similar to the corresponding fraction in fishes. Overall, we suggest that the manner in which the oxygen they require enters the body of small marine invertebrates, although often neglected, is a crucial aspect of their biology. In addition, based on our result that arrow worms conform to the GOLT, we suggest that this theory may provide the theoretical framework for the study of growth in the other water-breathing ectotherms lacking gills.

Quantitative mismatch between empirical temperature-size rule slopes and predictions based on oxygen limitation

In ectotherms, adult body size commonly declines with increasing environmental temperature, a pattern known as the temperature-size rule. One influential hypothesis explaining this observation is that the challenge of obtaining sufficient oxygen to support metabolism becomes greater with increasing body size, and more so at high temperatures. Yet, previous models based on this hypothesis do not account for phenotypic plasticity in the physiology of organisms that counteracts oxygen limitation at high temperature. Here, we model the predicted strength of the temperature-size response using estimates of how both the oxygen supply and demand is affected by temperature when allowing for phenotypic plasticity in the aquatic ectotherm Daphnia magna. Our predictions remain highly inconsistent with empirical temperature-size responses, with the prior being close to one order of magnitude stronger than the latter. These results fail to provide quantitative support for the hypothesis that oxygen limitation drives temperature-size clines in aquatic ectotherms. Future studies into the role of oxygen limitation should address how the strength of the temperature-size response may be shaped by evolution under fluctuating temperature regimes. Finally, our results caution against applying deterministic models based on the oxygen limitation hypothesis when predicting future changes in ectotherm size distributions under climate change.

Enhanced production of γ-amino acid 3-amino-4-hydroxybenzoic acid by recombinant Corynebacterium glutamicum under oxygen limitation

Background Bio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics. Results Under aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of < 33% saturated oxygen. Based on this metabolome analysis, metabolic pathways were rationally designed for oxygen limitation. An ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture. Conclusions Our results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives. Graphical Abstract

Quantitative Mismatch Between Empirical Temperature-Size Rule Slopes and Predictions Based on Oxygen Limitation

In ectotherms, adult body size commonly declines with increasing environmental temperature, a pattern known as the temperature-size rule. One influential hypothesis explaining this observation is that the challenge of obtaining sufficient oxygen to support metabolism becomes greater with increasing body size, and more so at high temperatures. Yet, previous models based on this hypothesis do not account for phenotypic plasticity in the physiology of organisms that counteracts oxygen limitation at high temperature. Here, we model the predicted strength of the temperature-size response using estimates of how both the oxygen supply and demand is affected by temperature when allowing for phenotypic plasticity in the aquatic ectotherm Daphnia magna. Our predictions remain highly inconsistent with empirical temperature-size responses, with the prior being close to one order of magnitude stronger than the latter. These results fail to provide quantitative support for the hypothesis that oxygen limitation drives temperature-size clines in aquatic ectotherms. Future studies into the role of oxygen limitation should address how the strength of the temperature-size response may be shaped by evolution under fluctuating temperature regimes. Finally, our results caution against applying deterministic models based on the oxygen limitation hypothesis when predicting future changes in ectotherm size distributions under climate change.

A universal microfluidic approach for quantitative study of bacterial biofilms

Bacteria usually live in densely packed communities called biofilms, where interactions between the bacteria give rise to complex properties. Quantitative analysis is indispensable in understanding those properties. However, current biofilm culturing approaches impose various limitations to these types of analysis. Here, we developed a microfluidic approach for quantitative study of biofilms, which is universal and can be used to culture biofilms of various bacterial species. To demonstrate the advantages of this approach, we present two examples, both of which revealed new biological insights. In the first example, we explored the response of Escherichia coli biofilms to exogenous hydrogen peroxide; We found the biofilms gained resistance to H2O2, but their growth was slowed down due to the metabolic cost of maintaining the resistance; However, under oxygen limitation, H2O2 can anti-intuitively boost biofilm growth. In the second example, we explored resource retention by Pseudomonas aeruginosa biofilms; We observed a fluorescent substance within the biofilm and identified it as the siderophore pyoverdine; We further showed that the extracellular matrix component Psl acted as a retention barrier for pyoverdine, minimizing its loss into the environment and therefore potentially promoting sharing of pyoverdine within the biofilm.

An Alanine Aminotransferase is Required for Polysaccharide Regulation and Resistance of Aspergillus fumigatus Biofilms to Echinocandin Treatment

Alanine metabolism has been suggested as an adaptation strategy to oxygen limitation in organisms ranging from plants to mammals. Within the pulmonary infection microenvironment A. fumigatus forms biofilms with steep oxygen gradients defined by regions of oxygen limitation. A significant increase in alanine levels was observed in A. fumigatus cultured under oxygen limiting conditions. An alanine aminotransferase, AlaA, was observed to function in alanine catabolism and is required for several aspects of A. fumigatus biofilm physiology. Loss of alaA, or its catalytic activity, results in decreased adherence of biofilms through a defect in the maturation of the extracellular matrix polysaccharide galactosaminogalactan (GAG). Additionally, exposure of cell wall polysaccharides is also impacted by loss of alaA and loss of AlaA catalytic activity confers increased biofilm susceptibility to echinocandin treatment which is correlated with enhanced fungicidal activity. The increase in echinocandin susceptibility is specific to biofilms and chemical inhibition of alaA by the alanine aminotransferase inhibitor β-chloro-L-alanine is sufficient to sensitize A. fumigatus biofilms to echinocandin treatment. Finally, loss of alaA increases susceptibility of A. fumigatus to in vivo echinocandin treatment in a murine model of invasive pulmonary aspergillosis. Our results provide insight into the interplay of metabolism, biofilm formation, and antifungal drug resistance in A. fumigatus and describes a mechanism of increasing susceptibility of A. fumigatus biofilms to the echinocandin class of antifungal drugs.

Glucose-Limited Fed-Batch Cultivation Strategy to Mimic Large-Scale Effects in Escherichia coli Linked to Accumulation of Non-Canonical Branched-Chain Amino Acids by Combination of Pyruvate Pulses and Dissolved Oxygen Limitation

Insufficient mixing in large-scale bioreactors provokes gradient zones of substrate, dissolved oxygen (DO), pH, and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and β-methylnorleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids (ncBCAA), but also that pyruvate pulses produce the same effect. Therefore, we combined the enzyme-based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor (STR). To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

Simultaneous anaerobic and aerobic ammonium and methane oxidation under oxygen limitation

Methane and ammonia have to be removed from wastewater treatment effluent in order to discharge it to receiving water bodies. A potential solution for this is a combination of simultaneous ammonia and methane oxidation by anaerobic ammonia-oxidizing (anammox) bacteria and nitrite/nitrate-dependent anaerobic methane oxidizing (N-damo) microorganisms. When applied, these microorganisms will be exposed to oxygen but little is known about the effect of a low concentration of oxygen on a culture containing these microorganisms. In this study, a stable co-culture containing anammox and N-damo microorganisms in a laboratory scale bioreactor was established under oxygen limitation. Membrane inlet mass spectrometry (MIMS) was used to directly measure the in situ simultaneous activity of N-damo, anammox and aerobic ammonia oxidizing microorganisms. In addition, batch tests revealed that the bioreactor also harbored aerobic methanotrophs and anaerobic methanogens. Together with FISH analysis and metagenomics, these results indicate that the combination of N-damo and anammox activity under the continuous supply of limiting oxygen concentrations is feasible and can be implemented for the removal of methane and ammonia from anaerobic digester effluents. Importance Nitrogen in wastewater leads to eutrophication of the receiving water bodies and methane is a potent greenhouse gas; it is therefore important that these are removed from wastewater. A potential solution for the simultaneous removal of nitrogenous compounds and methane is the application of a combination of nitrite/nitrate depended methane oxidation (N-damo) and anammox. In order to do so, it is important to investigate the effect of oxygen on these two anaerobic processes. In this study, we investigate the effect of a continuous oxygen supply on the activity of an anaerobic methane and ammonia oxidizing coculture. The findings presented in this study are important for the potential application of these two microbial processes in wastewater treatment.