Soil fluxomics analysis can provide pivotal information for understanding soil biochemical pathways and their regulation, but direct measurement methods are rare. Here, we describe an approach to measure soil extracellular metabolite (amino sugar and amino acid) concentrations and fluxes based on a ¹⁵N isotope pool dilution technique via liquid chromatography and high-resolution mass spectrometry. We produced commercially unavailable ¹⁵N and ¹³C labeled amino sugars and amino acids by hydrolyzing peptidoglycan isolated from isotopically labeled bacterial biomass and used them as tracers (¹⁵N) and internal standards (¹³C). High-resolution (Orbitrap Exactive) MS with a resolution of 50 000 allowed us to separate different stable isotope labeled analogues across a large range of metabolites. The utilization of ¹³C internal standards greatly improved the accuracy and reliability of absolute quantification. We successfully applied this method to two types of soils and quantified the extracellular gross fluxes of 2 amino sugars, 18 amino acids, and 4 amino acid enantiomers. Compared to the influx and efflux rates of most amino acids, similar ones were found for glucosamine, indicating that this amino sugar is released through peptidoglycan and chitin decomposition and serves as an important nitrogen source for soil microorganisms. d-Alanine and d-glutamic acid derived from peptidoglycan decomposition exhibited similar turnover rates as their l-enantiomers. This novel approach offers new strategies to advance our understanding of the production and transformation pathways of soil organic N metabolites, including the unknown contributions of peptidoglycan and chitin decomposition to soil organic N cycling.

Microbial nitrogen use efficiency (NUE) is the efficiency by which microbes allocate organic N acquired to biomass formation relative to the N in excess of microbial demand released through N mineralization. Microbial NUE thus is critical to estimate the capacity of soil microbes to retain N in soils and thereby affects inorganic N availability to plants and ecosystem N losses. However, how soil temperature and soil moisture/O₂ affect microbial NUE to date is not clear. Therefore, two independent incubation experiments were conducted with soils from three land uses (cropland, grassland and forest) on two bedrocks (silicate and limestone). Soils were exposed to 5, 15 and 25 °C overnight at 60% water holding capacity (WHC) or acclimated to 30 and 60% WHC at 21% O₂ and to 90% WHC at 1% O₂ over one week at 20 °C. Microbial NUE was measured as microbial growth over microbial organic N uptake (the sum of growth N demand and gross N mineralization). Microbial NUE responded positively to temperature increases with Q₁₀ values ranging from 1.30 ± 0.11 to 2.48 ± 0.67. This was due to exponentially increasing microbial growth rates with incubation temperature while gross N mineralization rates were relatively insensitive to temperature increases (Q₁₀ values 0.66 ± 0.30 to 1.63 ± 0.15). Under oxic conditions (21% O₂), microbial NUE as well as gross N mineralization were not stimulated by the increase in soil moisture from 30 to 60% WHC. Under suboxic conditions (90% WHC and 1% O₂), microbial NUE markedly declined as microbial growth rates were strongly negatively affected due to increasing microbial energy limitation. In contrast, gross N mineralization rates increased strongly as organic N uptake became in excess of microbial growth N demand. Therefore, in the moisture/O₂ experiment microbial NUE was mainly regulated by the shift in O₂ status (to suboxic conditions) and less affected by increasing water availability per se. These temperature and moisture/O₂ effects on microbial organic N metabolism were consistent across the soils differing in bedrock and land use. Overall it has been demonstrated that microbial NUE was controlled by microbial growth, and that NUE controlled gross N mineralization as an overflow metabolism when energy (C) became limiting or N in excess in soils. This study thereby greatly contributes to the understanding of short-term environmental responses of microbial community N metabolism and the regulation of microbial organic-inorganic N transformations in soils.

Amino sugars and D-amino acid enantiomers are major components of bacterial and fungal cell walls (i.e. peptidoglycan and chitin) and are often used as biomarkers of microbial residue turnover in soils. However, little is known about the in situ decomposition rates of microbial cell wall residues and how soil physicochemical properties affect this process. In this study, we investigated the in situ gross production and consumption rates of free amino sugars (glucosamine and muramic acid) and amino acids (meso-diaminopimelic acid, l-alanine, and d-alanine) by a novel isotope pool dilution assay using ¹⁵N-labeled amino compounds. Soils were obtained from six sites differing in land management (cropland, pasture, and forest) and bedrock (silicate and limestone) and incubated at three temperatures (5, 15, and 25 °C). Free glucosamine released during the decomposition of peptidoglycan and chitin contributed significantly to the extractable soil organic nitrogen pool. Gross production and consumption rates of glucosamine were higher than those of individual amino acids, i.e. L- and D-alanine. Muramic acid had a longer mean residence time (68 h compared to 2.7 h for glucosamine, L- and D-alanine) and made a negligible contribution to soil organic nitrogen fluxes, indicating that free muramic acid was not a major decomposition product of peptidoglycan in soils. Meso-diaminopimelic acid and D-alanine exhibited comparable gross production and consumption rates with L-alanine. These amino acids can be used as indicators to estimate the decomposition of peptidoglycan from bacterial cell wall residues. We found that chitin decomposition was greater in silicate soils, while peptidoglycan decomposition dominated in limestone soils. Glucosamine production rates were not correlated with soil total amino sugars, microbial community structure, or hydrolytic enzyme activities, but were highest in soils with low pH and high sand content, indicating that soil texture and soil pH may strongly influence the decomposition of amino sugar polymers. In contrast, mDAP, L- and D-alanine gross production and consumption rates were positively correlated with soil pH and clay content, due to greater depolymerization of peptidoglycan stem peptides in limestone soils. This isotope pool dilution approach strongly improves our understanding of the mechanisms and environmental controls on microbial cell wall decomposition in soils.

Proteins constitute the single largest soil organic nitrogen (SON) reservoir and its decomposition drives terrestrial N availability. Protein cleavage by extracellular enzymes is the rate limiting step in the soil organic N cycle and can be controlled by extracellular enzyme production or protein availability/stabilization in soil. Both controls can be affected by geology and land use, as well as be vulnerable to changes in soil temperature and moisture/O₂. To explore major controls of soil gross protein depolymerization we sampled six soils from two soil parent materials (calcareous and silicate), where each soil type included three land uses (cropland, pasture and forest). Soil samples were subjected to three temperature treatments (5, 15, 25 °C at 60% water-holding capacity (WHC) and aerobic conditions) or three soil moisture/O₂ treatments (30 and 60% WHC at 21% O₂, 90% WHC at 1% O₂, at 20 °C) in short-term experiments. Samples were incubated for one day in the temperature experiment and for one week in the moisture/O₂ experiment. Gross protein depolymerization rates were measured by a novel ¹⁵N isotope pool dilution approach. The low temperature sensitivity of gross protein depolymerization, the lack of relationship with protease activity and strong effects of soil texture and pH demonstrate that this process is constrained by organo-mineral associations and not by soil enzyme content. This also became apparent from the inverse effects in calcareous and silicate soils caused by water saturation/O₂ limitation. We highlight that the specific soil mineralogy influenced the response of gross depolymerization rates to water saturation/O₂ limitation, causing (I) increasing gross depolymerization rates due to release of adsorbed proteins by reductive dissolution of Fe- and Mn-oxyhydroxides in calcareous soils and (II) decreasing gross depolymerization rates due to mobilization of coagulating and toxic Al³⁺ compounds in silicate soils.

The ratio of carbon (C) that is invested into microbial growth to organic C taken up is known as microbial carbon use efficiency (CUE), which is influenced by environmental factors such as soil temperature and soil moisture. How microbes will physiologically react to short-term environmental changes is not well understood, primarily due to methodological restrictions. Here we report on two independent laboratory experiments to explore short-term temperature and soil moisture effects on soil microbial physiology (i.e. respiration, growth, CUE, and microbial biomass turnover): (i) a temperature experiment with 1-day pre-incubation at 5, 15 and 25 °C at 60% water holding capacity (WHC), and (ii) a soil moisture/oxygen (O₂) experiment with 7-day pre-incubation at 20 °C at 30%, 60% WHC (both at 21% O₂) and 90% WHC at 1% O₂. Experiments were conducted with soils from arable, pasture and forest sites derived from both silicate and limestone bedrocks. We found that microbial CUE responded heterogeneously though overall positively to short-term temperature changes, and decreased significantly under high moisture level (90% WHC)/suboxic conditions due to strong decreases in microbial growth. Microbial biomass turnover time decreased dramatically with increasing temperature, and increased significantly at high moisture level (90% WHC)/suboxic conditions. Our findings reveal that the responses of microbial CUE and microbial biomass turnover to short-term temperature and moisture/O₂ changes depended mainly on microbial growth responses and less on respiration responses to the environmental cues, which were consistent across soils differing in land use and geology.

Species-rich plant communities have been shown to be more productive and to exhibit increased long-term soil organic carbon (SOC) storage. Soil microorganisms are central to the conversion of plant organic matter into SOC, yet the relationship between plant diversity, soil microbial growth, turnover as well as carbon use efficiency (CUE) and SOC accumulation is unknown. As heterotrophic soil microbes are primarily carbon limited, it is important to understand how they respond to increased plant-derived carbon inputs at higher plant species richness (PSR). We used the long-term grassland biodiversity experiment in Jena, Germany, to examine how microbial physiology responds to changes in plant diversity and how this affects SOC content. The Jena Experiment considers different numbers of species (1-60), functional groups (1-4) as well as functional identity (small herbs, tall herbs, grasses, and legumes). We found that PSR accelerated microbial growth and turnover and increased microbial biomass and necromass. PSR also accelerated microbial respiration, but this effect was less strong than for microbial growth. In contrast, PSR did not affect microbial CUE or biomass-specific respiration. Structural equation models revealed that PSR had direct positive effects on root biomass, and thereby on microbial growth and microbial biomass carbon. Finally, PSR increased SOC content via its positive influence on microbial biomass carbon. We suggest that PSR favors faster rates of microbial growth and turnover, likely due to greater plant productivity, resulting in higher amounts of microbial biomass and necromass that translate into the observed increase in SOC. We thus identify the microbial mechanism linking species-rich plant communities to a carbon cycle process of importance to Earth's climate system.

Biocrusts are phototroph-driven communities inhabiting arid soil surfaces. Like plants, most photoautotrophs (largely cyanobacteria) in biocrusts are thought to exchange fixed carbon for essential nutrients like nitrogen with cyanosphere bacteria. Here, we aim to compare beneficial interactions in rhizosphere and cyanosphere environments, including finding growth-promoting strains for hosts from both environments. To examine this, we performed a retrospective analysis of 16S rRNA gene sequencing datasets, host-microbe co-culture experiments between biocrust communities/biocrust isolates and a model grass (Brachypodium distachyon) or a dominant biocrust cyanobacterium (Microcoleus vaginatus), and metabolomic analysis. All 18 microbial phyla in the cyanosphere were also present in the rhizosphere, with additional 17 phyla uniquely found in the rhizosphere. The biocrust microbes promoted the growth of the model grass, and three biocrust isolates (Bosea sp._L1B56, Pseudarthrobacter sp._L1D14 and Pseudarthrobacter picheli_L1D33) significantly promoted the growth of both hosts. Moreover, pantothenic acid was produced by Pseudarthrobacter sp._L1D14 when grown on B. distachyon exudates, and supplementation of plant growth medium with this metabolite increased B. distachyon biomass by over 60%. These findings suggest that cyanobacteria and other diverse photoautotrophic hosts can be a source for new plant growth-promoting microbes and metabolites.

Microorganisms are critical in mediating carbon (C) and nitrogen (N) cycling processes in soils. Yet, it has long been debated whether the processes underlying biogeochemical cycles are affected by the composition and diversity of the soil microbial community or not. The composition and diversity of soil microbial communities can be influenced by various environmental factors, which in turn are known to impact biogeochemical processes. The objectives of this study were to test effects of multiple edaphic drivers individually and represented as the multivariate soil environment interacting with microbial community composition and diversity, and concomitantly on multiple soil functions (i.e. soil enzyme activities, soil C and N processes). We employed high-throughput sequencing (Illumina MiSeq) to analyze bacterial/archaeal and fungal community composition by targeting the 16S rRNA gene and the ITS1 region of soils collected from three land uses (cropland, grassland and forest) deriving from two bedrock forms (silicate and limestone). Based on this data set we explored single and combined effects of edaphic variables on soil microbial community structure and diversity, as well as on soil enzyme activities and several soil C and N processes. We found that both bacterial/archaeal and fungal communities were shaped by the same edaphic factors, with most single edaphic variables and the combined soil environment representation exerting stronger effects on bacterial/archaeal communities than on fungal communities, as demonstrated by (partial) Mantel tests. We also found similar edaphic controls on the bacterial/archaeal/fungal richness and diversity. Soil C processes were only directly affected by the soil environment but not affected by microbial community composition. In contrast, soil N processes were significantly related to bacterial/archaeal community composition and bacterial/archaeal/fungal richness/diversity but not directly affected by the soil environment. This indicates direct control of the soil environment on soil C processes and indirect control of the soil environment on soil N processes by structuring the microbial communities. The study further highlights the importance of edaphic drivers and microbial communities (i.e. composition and diversity) on important soil C and N processes.

Purpose

To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats.

Methods

Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity.

Results

Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity.

Conclusions

These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

Background and objective

To develop a safe and efficient surgical procedure for subretinal implantation into porcine eyes of a human embryonic stem cell-derived retinal pigmented epithelium (hESC-RPE) monolayer seeded onto a Parylene-C scaffold. This implant is referred to as CPCB-RPE1.

Materials and methods

Ultrathin Parylene-C scaffolds were seeded with hESC-RPE and surgically implanted into the subretinal space of Yucatan mini pigs (n = 8). The surgery consisted of pars plana vitrectomy, induction of a limited retinal detachment, and peripheral retinotomy for insertion of the monolayer using a novel tissue injector, followed by silicone oil tamponade injection, laser photocoagulation around the retinotomy site, and inferior iridectomy. Oral cyclosporine was administered from day 1 and during the entire follow-up period. Three months later, the animals were euthanized and the eyes and major organs were submitted for histological analysis. Adjacent sections underwent immunohistochemical analysis to detect human cells using anti-TRA-1-85 (human blood group antigen) antibody and DAPI antibodies.

Results

The cell monolayer was immunopositive for TRA-1-85 3 months after implantation and migration from the Parylene-C scaffold was not detected. One eye had a mild inflammatory reaction around the implant that was negative for human biomarkers. No intraocular or systemic tumors were detected.

Conclusion

The hESC-RPE cells survived for 3 months in this animal model. The surgical procedure for subretinal implantation of CPCB-RPE1 is feasible and safe, without cell migration off the scaffold or development of ocular or systemic tumors.