Protein production dynamics and physiological adaptation of recombinant Komagataella phaffii at near-zero growth rates.

BACKGROUND: Specific productivity (qP) in yeast correlates with growth, typically peaking at intermediate or maximum specific growth rates (µ). Understanding the factors limiting productivity at extremely low µ might reveal decoupling strategies, but knowledge of production dynamics and physiology in such conditions is scarce. Retentostats, a type of continuous cultivation, enable the well-controlled transition to near-zero µ through the combined retention of biomass and limited substrate supply. Recombinant Komagataella phaffii (syn Pichia pastoris) secreting a bivalent single domain antibody (VHH) was cultivated in aerobic, glucose-limited retentostats to investigate recombinant protein production dynamics and broaden our understanding of relevant physiological adaptations at near-zero growth conditions. RESULTS: By the end of the retentostat cultivation, doubling times of approx. two months were reached, corresponding to µ = 0.00047 h-1. Despite these extremely slow growth rates, the proportion of viable cells remained high, and de novo synthesis and secretion of the VHH were observed. The average qP at the end of the retentostat was estimated at 0.019 mg g-1 h-1. Transcriptomics indicated that genes involved in protein biosynthesis were only moderately downregulated towards zero growth, while secretory pathway genes were mostly regulated in a manner seemingly detrimental to protein secretion. Adaptation to near-zero growth conditions of recombinant K. phaffii resulted in significant changes in the total protein, RNA, DNA and lipid content, and lipidomics revealed a complex adaptation pattern regarding the lipid class composition. The higher abundance of storage lipids as well as storage carbohydrates indicates that the cells are preparing for long-term survival. CONCLUSIONS: In conclusion, retentostat cultivation proved to be a valuable tool to identify potential engineering targets to decouple growth and protein production and gain important insights into the physiological adaptation of K. phaffii to near-zero growth conditions.

Comparative Analysis of Various Spider Silks in Regard to Nerve Regeneration: Material Properties and Schwann Cell Response.

Peripheral nerve reconstruction through the employment of nerve guidance conduits with Trichonephila dragline silk as a luminal filling has emerged as an outstanding preclinical alternative to avoid nerve autografts. Yet, it remains unknown whether the outcome is similar for silk fibers harvested from other spider species. This study compares the regenerative potential of dragline silk from two orb-weaving spiders, Trichonephila inaurata and Nuctenea umbratica, as well as the silk of the jumping spider Phidippus regius. Proliferation, migration, and transcriptomic state of Schwann cells seeded on these silks are investigated. In addition, fiber morphology, primary protein structure, and mechanical properties are studied. The results demonstrate that the increased velocity of Schwann cells on Phidippus regius fibers can be primarily attributed to the interplay between the silk's primary protein structure and its mechanical properties. Furthermore, the capacity of silk fibers to trigger cells toward a gene expression profile of a myelinating Schwann cell phenotype is shown. The findings for the first time allow an in-depth comparison of the specific cellular response to various native spider silks and a correlation with the fibers' material properties. This knowledge is essential to open up possibilities for targeted manufacturing of synthetic nervous tissue replacement.

Recycling nitrogen from liquid digestate via novel reactive struvite and zeolite minerals to mitigate agricultural pollution.

Recycling nutrients is of paramount importance. For this reason, struvite and nitrogen enriched zeolite fertilizers produced from wastewater treatments are receiving growing attention in European markets. However, their effects on agricultural soils are far from certain, especially struvite, which only recently was implemented in EU Fertilizing Product Regulations. In this paper, we investigate the effects of these materials in acid sandy arable soil, particularly focusing on N dynamics, evaluating potential losses, transformation pathways, and the effects of struvite and zeolitic tuffs on main soil biogeochemical parameters, in comparison to traditional fertilization with digestate. Liming effect (pH alkalinization) was observed in all treatments with varying intensities, affecting most of the soil processes. The struvite was quickly solubilized due to soil acidity, and the release of nutrients stimulated nitrifying and denitrifying microorganisms. Zeolitic tuff amendments decreased the NOx gas emissions, which are precursors to the powerful climate altering N2O gas, and the N enriched chabazite tuff also recorded smaller NH3 emissions compared to the digestate. However, a high dosage of zeolites in soil increased NH3 emissions after fertilization, due to pronounced pH shifts. Contrasting effects were observed between the two zeolitic tuffs when applied as soil amendments; while the chabazite tuff had a strong positive effect - increasing up to ∼90% the soil microbial N immobilization - the employed clinoptilolite tuff had immediate negative effects on the microbial biomass, likely due to the large quantities of sulphur released. However, when applied at lower dosages, the N enriched clinoptilolite also contributed to the increase of microbial N. From these outcomes, we confirm the potential of struvite and zeolites to mitigate the outfluxes of nutrients from agricultural systems. To gain the best results and significantly lower environmental impacts, extension practitioners could give recommendations based on the soils that are planned for zeolite application.

Soil and biomass carbon re-accumulation after landslide disturbances.

In high-standing islands of the Western Pacific, typhoon-triggered landslides occasionally strip parts of the landscape of its vegetative cover and soil layer and export large amounts of biomass and soil organic carbon (OC) from land to the ocean. After such disturbances, new vegetation colonizes the landslide scars and OC starts to reaccumulate. In the subtropical mountains of Taiwan and in other parts of the world, bamboo (Bambusoideae) species may invade at a certain point in the succession of recovering landslide scars. Bamboo has a high potential for carbon sequestration because of its fast growth and dense rooting system. However, it is still largely unknown how these properties translate into soil OC re-accumulation rates after landslide disturbance. In this study, a chronosequence was established on four former landslide scars in the Central Mountain Range of Taiwan, ranging in age from 6 to 41 years post disturbance as determined by landslide mapping from remote sensing. The younger landslide scars were colonized by Miscanthus floridulus, while after approx. 15 to 20 years of succession, bamboo species (Phyllostachys) were dominating. Biomass and soil OC stocks were measured on the recovering landslide scars and compared to an undisturbed Cryptomeria japonica forest stand in the area. After initially slow re-vegetation, biomass carbon accumulated in Miscanthus stands with mean annual accretion rates of 2 ± 0.5 Mg C ha-1 yr-1. Biomass carbon continued to increase after bamboo invasion and reached ~40% of that in the reference forest site after 41 years of landslide recovery. Soil OC accumulation rates were ~2.0 Mg C ha-1 yr-1, 6 to 41 years post disturbance reaching ~64% of the level in the reference forest. Our results from this in-situ study suggest that recovering landslide scars are strong carbon sinks once an initial lag period of vegetation re-establishment is overcome.

Fungicide application increased copper-bioavailability and impaired nitrogen fixation through reduced root nodule formation on alfalfa.

Copper-based fungicides have been used for a long time in viticulture and have accumulated in many vineyard soils. In this study, incrementing Cu(OH)2-based fungicide application from 0.05 to 5 g Cu kg-1 on two agricultural soils (an acidic sandy loam (L, pH 4.95) and an alkaline silt loam (D, pH 7.45)) resulted in 5 times more mobile Cu in the acidic soil. The most sensitive parameters of alfalfa (Medicago sativa) growing in these soils were the root nodule number, decreasing to 34% and 15% of the control at 0.1 g Cu kg-1 in soil L and at 1.5 g Cu kg-1 in soil D, respectively, as well as the nodule biomass, decreasing to 25% and 27% at 0.5 g Cu kg-1 in soil L and at 1.5 g Cu kg-1 in soil D, respectively. However, the enzymatic N2-fixation was not directly affected by Cu in spite of the presence of Cu in the meristem and the zone of effective N2-fixation, as illustrated by chemical imaging. The strongly different responses observed in the two tested soils reflect the higher buffering capacity of the alkaline silt loam and showed that Cu mitigation and remediation strategies should especially target vineyards with acidic, sandy soils.

Biochar application increases sorption of nitrification inhibitor 3,4-dimethylpyrazole phosphate in soil.

Biochar (BC) application to soils is of growing interest as a strategy to improve soil fertility and mitigate climate change. However, BC-induced alterations in the soil N cycle are currently under debate. BC has recently been shown to accelerate the emissions of N2O via the biotic ammonium oxidation pathway, which results in lower nitrogen use efficiency and environmentally harmful losses of NO3 and/ or N2O. To avoid these potential losses, the use of nitrification inhibitor (NI) could provide a useful mitigation strategy for BC-amended agricultural fields. Here, we tested the sorption behavior of a model NI, the synthetic 3,4-dimethylpyrazole phosphate (DMPP) on 15-month-aged soil-BC mixtures. We saw that BC additions increased DMPP sorption to varying extents depending on BC feedstock type and pyrolysis temperature. The highest sorption was found for BC pyrolyzed at a lower temperature. BC effects on soil physico-chemical characteristics (i.e., hydrophobicity) seem to be important factors.

Assessment of Cu applications in two contrasting soils-effects on soil microbial activity and the fungal community structure.

Copper (Cu)-based fungicides have been used in viticulture to prevent downy mildew since the end of the 19th century, and are still used today to reduce fungal diseases. Consequently, Cu has built up in many vineyard soils, and it is still unclear how this affects soil functioning. The present study aimed to assess the short and medium-term effects of Cu contamination on the soil fungal community. Two contrasting agricultural soils, an acidic sandy loam and an alkaline silt loam, were used for an eco-toxicological greenhouse pot experiment. The soils were spiked with a Cu-based fungicide in seven concentrations (0-5000 mg Cu kg-1 soil) and alfalfa was grown in the pots for 3 months. Sampling was conducted at the beginning and at the end of the study period to test Cu toxicity effects on total microbial biomass, basal respiration and enzyme activities. Fungal abundance was analysed by ergosterol at both samplings, and for the second sampling, fungal community structure was evaluated via ITS amplicon sequences. Soil microbial biomass C as well as microbial respiration rate decreased with increasing Cu concentrations, with EC50 ranging from 76 to 187 mg EDTA-extractable Cu kg-1 soil. Oxidative enzymes showed a trend of increasing activity at the first sampling, but a decline in peroxidase activity was observed for the second sampling. We found remarkable Cu-induced changes in fungal community abundance (EC50 ranging from 9.2 to 94 mg EDTA-extractable Cu kg-1 soil) and composition, but not in diversity. A large number of diverse fungi were able to thrive under elevated Cu concentrations, though within the order of Hypocreales several species declined. A remarkable Cu-induced change in the community composition was found, which depended on the soil properties and, hence, on Cu availability.

Non-target effects of a glyphosate-based herbicide on Common toad larvae (Bufo bufo, Amphibia) and associated algae are altered by temperature.

BACKGROUND: Glyphosate-based herbicides are the most widely used pesticides in agriculture, horticulture, municipalities and private gardens that can potentially contaminate nearby water bodies inhabited by amphibians and algae. Moreover, the development and diversity of these aquatic organisms could also be affected by human-induced climate change that might lead to more periods with extreme temperatures. However, to what extent non-target effects of these herbicides on amphibians or algae are altered by varying temperature is not well known. METHODS: We studied effects of five concentrations of the glyphosate-based herbicide formulation Roundup PowerFlex (0, 1.5, 3, 4 mg acid equivalent glyphosate L-1 as a one time addition and a pulse treatment of totally 4 mg a.e. glyphosate L-1) on larval development of Common toads (Bufo bufo, L.; Amphibia: Anura) and associated algae communities under two temperature regimes (15 vs. 20 °C). RESULTS: Herbicide contamination reduced tail growth (-8%), induced the occurrence of tail deformations (i.e. lacerated or crooked tails) and reduced algae diversity (-6%). Higher water temperature increased tadpole growth (tail and body length (tl/bl) +66%, length-to-width ratio +4%) and decreased algae diversity (-21%). No clear relation between herbicide concentrations and tadpole growth or algae density or diversity was observed. Interactive effects of herbicides and temperature affected growth parameters, tail deformation and tadpole mortality indicating that the herbicide effects are temperature-dependent. Remarkably, herbicide-temperature interactions resulted in deformed tails in 34% of all herbicide treated tadpoles at 15 °C whereas no tail deformations were observed for the herbicide-free control at 15 °C or any tadpole at 20 °C; herbicide-induced mortality was higher at 15 °C but lower at 20 °C. DISCUSSION: These herbicide- and temperature-induced changes may have decided effects on ecological interactions in freshwater ecosystems. Although no clear dose-response effect was seen, the presence of glyphosate was decisive for an effect, suggesting that the lowest observed effect concentration (LOEC) in our study was 1.5 mg a.e. glyphosate L-1 water. Overall, our findings also question the relevance of pesticide risk assessments conducted at standard temperatures.

Erratum to: Study of soil aggregate breakdown dynamics under low dispersive ultrasonic energies with sedimentation and X-ray attenuation.

[This corrects the article PMC4834993.].

Study of soil aggregate breakdown dynamics under low dispersive ultrasonic energies with sedimentation and X-ray attenuation.

It has been increasingly recognized that soil organic matter stabilization is strongly controlled by physical binding within soil aggregates. It is therefore essential to measure soil aggregate stability reliably over a wide range of disruptive energies and different aggregate sizes. To this end, we tested high-accuracy ultrasonic dispersion in combination with subsequent sedimentation and X-ray attenuation. Three arable topsoils (notillage) from Central Europe were subjected to ultrasound at four different specific energy levels: 0.5, 6.7, 100 and 500 J cm-3, and the resulting suspensions were analyzed for aggregate size distribution by wet sieving (2 000-63 µm) and sedimentation/X-ray attenuation (63-2 µm). The combination of wet sieving and sedimentation technique allowed for a continuous analysis, at high resolution, of soil aggregate breakdown dynamics after defined energy inputs. Our results show that aggregate size distribution strongly varied with sonication energy input and soil type. The strongest effects were observed in the range of low specific energies (< 10 J cm-3), which previous studies have largely neglected. This shows that low ultrasonic energies are required to capture the full range of aggregate stability and release of soil organic matter upon aggregate breakdown.

Determination of organic and inorganic carbon in forest soil samples by mid-infrared spectroscopy and partial least squares regression.

Analyses of organic and inorganic carbon are of great interest in the field of soil analyses. Soil samples from a national monitoring project were provided for this study, including more than 130 forest sites from Austria. We investigated the humus layers (if present undecomposed litter (L), of mixed samples of F- (intermediate decomposed organic matter) and H-(highly decomposed organic matter) (FH)) and upper mineral soil layers (0-5 and 5-10 cm) of the samples. Mid-infrared spectra were recorded and evaluated by their band areas; subsequently we calculated models with the partial least squares approach. This was done by correlating calculated data of the mid-infrared spectra with gas-volumetrically determined carbonate values and measurements of organic carbon from an elemental analyzer. For carbonate determination, this approach gave satisfying results. For measurements of organic carbon, it was necessary to discriminate into humus layers and mineral soils or even more groups to obtain satisfactory correlations between spectroscopically determined and conventionally measured values. These additional factors were the presence of carbonate, the forest type, and the dominant tree species. In mineral soils, fewer subdivisions were necessary to obtain useful results. In humus layers, groupings of sites with more similar characteristics had to be formed in order to obtain satisfying results. The conclusion is that the chemical background of soil organic matter leading to different proportions of functional groups, especially in the less humified organic matter of the humus layers, plays a key role in analyses with mid-infrared spectroscopy. Keeping this in mind, the present approach has a significant potential for the prediction of properties of forest soil layers, such as, e.g., carbonate and organic carbon contents.

Determination of glyphosate and AMPA in surface and waste water using high-performance ion chromatography coupled to inductively coupled plasma dynamic reaction cell mass spectrometry (HPIC-ICP-DRC-MS).

A novel method employing high-performance cation chromatography in combination with inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS) for the simultaneous determination of the herbicide glyphosate (N-phosphonomethylglycine) and its main metabolite aminomethyl phosphonic acid (AMPA) is presented. P was measured as (31)P(16)O(+) using oxygen as reaction gas. For monitoring the stringent target value of 0.1 µg L(-1) for glyphosate, applicable for drinking and surface water within the EU, a two-step enrichment procedure employing Chelex 100 and AG1-X8 resins was applied prior to HPIC-ICP-MS analysis. The presented approach was validated for surface water, revealing concentrations of 0.67 µg L(-1) glyphosate and 2.8 µg L(-1) AMPA in selected Austrian river water samples. Moreover, investigations at three waste water-treatment plants showed that elimination of the compounds at the present concentration levels was not straightforward. On the contrary, all investigated plant effluents showed significant amounts of both compounds. Concentration levels ranged from 0.5-2 µg L(-1) and 4-14 µg L(-1) for glyphosate and AMPA, respectively.