Multi-organ single-cell RNA sequencing in mice reveals early hyperglycemia responses that converge on fibroblast dysregulation.

Diabetes causes a range of complications that can affect multiple organs. Hyperglycemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis, and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycemia across multiple tissues using single-cell RNA sequencing (scRNA-seq) and identified conserved, as well as organ-specific, changes associated with diabetes complications. By studying an early time point of diabetes, we focus on biological processes involved in the initiation of the disease, before the later organ-specific manifestations had supervened. We used a mouse model of type 1 diabetes and performed scRNA-seq on cells isolated from the heart, kidney, liver, and spleen of streptozotocin-treated and control male mice after 8 weeks and assessed differences in cell abundance, gene expression, pathway activation, and cell signaling across organs and within organs. In response to hyperglycemia, endothelial cells, macrophages, and monocytes displayed organ-specific transcriptional responses, whereas fibroblasts showed similar responses across organs, exhibiting altered metabolic gene expression and increased myeloid-like fibroblasts. Furthermore, we found evidence of endothelial dysfunction in the kidney, and of endothelial-to-mesenchymal transition in streptozotocin-treated mouse organs. In summary, our study represents the first single-cell and multi-organ analysis of early dysfunction in type 1 diabetes-associated hyperglycemia, and our large-scale dataset (comprising 67 611 cells) will serve as a starting point, reference atlas, and resource for further investigating the events leading to early diabetic disease.

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions.

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell-derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and identified conditions for the robust and unlimited expansion of hPS cell-derived PP cells under GMP-compliant conditions through a hypothesis-driven iterative approach. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling, and inhibition of selected branches of the TGFß and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over 10 passages and 40-45 d, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cell lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant ß-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell-derived PP cells. They will enable the establishment of large banks of GMP-produced PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine, and cell therapies.

Impact of PVC microplastics on soil chemical and microbiological parameters.

Microplastics (MPs) are emerging pollutants whose occurrence is a global problem in natural ecosystems including soil. Among MPs, polyvinyl chloride (PVC) is a well-known polymer with remarkable resistance to degradation, and because its recalcitrant nature serious environmental concerns are created during manufacturing and waste disposal. The effect of PVC (0.021% w/w) on chemical and microbial parameters of an agricultural soil was tested by a microcosm experiment at different incubation times (from 3 to 360 days). Among chemical parameters, soil CO2 emission, fluorescein diacetate (FDA) activity, total organic C (TOC), total N, water extractable organic C (WEOC), water extractable N (WEN) and SUVA254 were considered, while the structure of soil microbial communities was studied at different taxonomic levels (phylum and genus) by sequencing bacterial 16S and fungal ITS2 rDNA (Illumina MiSeq). Although some fluctuations were found, chemical and microbiological parameters exhibited some significant trends. Significant (p < 0.05) variations of soil CO2 emission, FDA hydrolysis, TOC, WEOC and WEN were found in PVC-treated soils over different incubation times. Considering the structure of soil microbial communities, the presence of PVC significantly (p < 0.05) affected the abundances of specific bacterial and fungal taxa: Candidatus_Saccharibacteria, Proteobacteria, Actinobacteria, Acidobacteria and Bacteroides among bacteria, and Basidiomycota, Mortierellomycota and Ascomycota among fungi. After one year of experiment, a reduction of the number and the dimensions of PVC was detected supposing a possible role of microorganisms on PVC degradation. The abundance of both bacterial and fungal taxa at phylum and genus level was also affected by PVC, suggesting that the impact of this polymer could be taxa-dependent.

Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.

AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Environmental consequences of the treatment of corn contaminated by aflatoxin B1 with co-digestion and co-composting in a life cycle perspective.

Global environmental performances of anaerobic co-digestion and co-composting of aflatoxin B1 (AFB1) contaminated corn were investigated by a life cycle assessment approach. Anaerobic co-digestion of pig slurry and corn with 25 µgkg-1 ww AFB1 concentration resulted able to generate 627 NLkgVS-1 of biogas with a reduction of the AFB1 concentration in the digestate of 44%. At AFB1 concentration of 100 µg kg-1 ww, the process resulted strongly inhibited with a biogas generation of 122 NLkgVS-1 and AFB1 concentration reduction in the digestate of 25%. Co-composting of 100 µg kg-1 dw AFB1 contaminated corn with other substrates as organic fraction of municipal waste, pig slurry, and other lignin-cellulosic residues showed a removal efficiency of AFB1 ranging from about 80 up to 95% depending on the different mixtures adopted. Environmental consequences associated to the removal of 1 mg of AFB1 in different scenarios investigated, including also the use on land of the digestate and of the compost, indicated that global warming was affected equally by co-digestion and co-composting, about 95 kgCO2eq. Co-digestion showed also the possibility of achieving avoided emissions of about - 0.007 kgNMVOCeq, - 2.5E-3 kgPeq, and - 30CTUe. Benefits concerning resource depletion resulted higher for co-composting due to the high amount of mineral fertilizer replaced. Contribution of AFB1 in the determination of human health (DALY) resulted lower than about 4% for co-digestion and practically negligible for co-composting.

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair.

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.

Benefits and risks of long-term recycling of pharmaceutical sewage sludge on agricultural soil.

European policy is direct towards increasing the agricultural reuse of sludge on soil for improving the fertility; however, the effects of long-term pharmaceutical sewage sludge (PSS) application on soil properties are still unknown. Thus, the aim of this work was to evaluate the agronomic and environmental effects on soil after 17 years of organic amendment with PSS derived from daptomycin production. Five different doses of PSS were spread on lands located in Anagni, Central Italy. Physico-chemical soil properties were investigated, as well as total and bioavailable heavy metals, changes in the soil organic matter quality and biochemical functioning. PSS application showed a positive agronomic potential, improving SOM quality, increasing soil humified organic matter and raising plant nutrients. SOM dynamic was different at low and high PSS supplies, as confirmed by the chemical and biochemical analysis (e.g. C biomass, FDA hydrolysis activity, basal respiration, dehydrogenase, urease and phosphatase activities). However, in a long-term agricultural reuse, environmental risks of PSS recycling were related to the increase of some heavy metals (Hg, Zn and Cu) and exchangeable Na.

Protein Methyltransferase Inhibition Decreases Endocrine Specification Through the Upregulation of Aldh1b1 Expression.

Understanding the mechanisms that promote the specification of pancreas progenitors and regulate their self-renewal and differentiation will help to maintain and expand pancreas progenitor cells derived from human pluripotent stem (hPS) cells. This will improve the efficiency of current differentiation protocols of hPS cells into ß-cells and bring such cells closer to clinical applications for the therapy of diabetes. Aldehyde dehydrogenase 1b1 (Aldh1b1) is a mitochondrial enzyme expressed specifically in progenitor cells during mouse pancreas development, and we have shown that its functional inactivation leads to accelerated differentiation and deficient ß-cells. In this report, we aimed to identify small molecule inducers of Aldh1b1 expression taking advantage of a mouse embryonic stem (mES) cell Aldh1b1 lacZ reporter line and a pancreas differentiation protocol directing mES cells into pancreatic progenitors. We identified AMI-5, a protein methyltransferase inhibitor, as an Aldh1b1 inducer and showed that it can maintain Aldh1b1 expression in embryonic pancreas explants. This led to a selective reduction in endocrine specification. This effect was due to a downregulation of Ngn3, and it was mediated through Aldh1b1 since the effect was abolished in Aldh1b1 null pancreata. The findings implicated methyltransferase activity in the regulation of endocrine differentiation and showed that methyltransferases can act through specific regulators during pancreas differentiation. Stem Cells 2019;37:640-651.

Effect of the mycotoxin aflatoxin B1 on a semi-continuous anaerobic digestion process.

Cereals are primary crops and are the most important raw material for feed and food production. Increasing aflatoxin B1 (AFB1) contamination of corn is an emerging issue, and disposal procedures for AFB1-contaminated corn are not currently defined. Recovery of contaminated corn through anaerobic digestion may represent a suitable strategy for its valorisation; however, only a few studies concerning the effect of AFB1 on anaerobic processes can be found. Thus, the purpose of the present work was to evaluate the effect of the mycotoxin AFB1 on a semi-continuous anaerobic digestion (AD) process. Semi-continuous trials were carried out, and the biomethane production from ABF1-contaminated feedstocks (25, 50, and 100 µg kg-1 AFB1 wet weight) was compared to that from non-contaminated feedstock. Moreover, the feasibility of the agronomic re-use of the digestate, and the fate of AFB1 during AD was assessed. No adverse effect of 25 µg kg-1 AFB1 contamination of feedstock on biomethane yield was observed. In contrast, 100 µg kg-1 AFB1 in the feedstock resulted in inhibition of the process due to the accumulation of organic acids, and to the decrease of the pH in the digestate (from 8.1 to 5.4). The continuous addition of AFB1-contaminated feedstock led to accumulation of the mycotoxin in the digestates. Consequently, a composting process should always precede the agricultural re-use of digestates in order to remove AFB1 and the residual phytotoxicity.

Evaluation of benefits and risks associated with the agricultural use of organic wastes of pharmaceutical origin.

Industrial fermentations for the production of pharmaceuticals generate large volumes of wastewater that can be biologically treated to recover plant nutrients through the application of pharmaceutical-derived wastes to the soil. Nevertheless, benefits and risks associated with their recovery are still unexplored. Thus, the aim of the present work was to characterize three potential organic residues (sludge, anaerobic digestate and compost) derived from the wastewater generated by the daptomycin production process. The main parameters evaluated were the physico-chemical properties, potential contaminants (heavy metals, pathogens and daptomycin residues), organic matter stabilization and the potential toxicity towards soil microorganisms and plants. The results showed that all the studied materials were characterized by high concentrations of plant macronutrients (N, P and K), making them suitable for agricultural reuse. Heavy metal contents and pathogens were under the limits established by European and Italian legislations, avoiding the risk of soil contamination. The compost showed the highest organic matter stabilization within the studied materials, whereas the sludge and the anaerobic digestate were characterized by large amounts of labile organic compounds. Although the pharmaceutical-derived fertilizers did not negatively affect the soil microorganisms, as demonstrated by the enzymatic activities, the sludge and the anaerobic digestate caused a moderate and strong phytotoxicity, respectively. The compost showed no toxic effect towards plant development and, moreover, it positively affected the germination and growth in lettuce and barley. The results obtained in the present study demonstrate that the valorization of pharmaceutical-derived materials through composting permits their agricultural reuse and also represents a suitable strategy to move towards a zero-waste production process for daptomycin.

Valorization of a pharmaceutical organic sludge through different composting treatments.

Nowadays, the agricultural reuse of pharmaceutical sludge is still limited due to environmental and agronomic issues (e.g. low stabilization of the organic matter, phytotoxicity). The aim of the present study was to evaluate the characteristics of a pharmaceutical sludge derived from the daptomycin production and to study the possibility of improving its quality through composting. The pharmaceutical sludge showed high content of macronutrients (e.g. total Kjeldahl N content was 38 g kg-1), but it was also characterized by high salinity (7.9 dS m-1), phytotoxicity (germination index was 36.7%) and a low organic matter stabilization. Two different mixtures were prepared (mixture A: 70% sludge + 30% wood chips w/w, mixture B: 45% sludge + 45% wood chips + 10% cereal straw w/w) and treated through static composting using two different aeration systems: active and passive aeration. The mixtures resulted in the production of two different compost, and the evolution of process management parameters was different. The low total solids and organic matter content of mixture A led to the failure of the process. The addition of cereal straw in mixture B resulted in increased porosity and C/N ratio and, consequently, in an optimal development of the composting process (e.g. the final organic matter loss was 54.1% and 63.1% for the passively and actively aerated treatment, respectively). Both passively and actively aerated composting of mixture B improved the quality of the pharmaceutical sludge, by increasing its organic matter stabilization and removing phytotoxicity.

Multi-approach characterization of organic sediment produced by an anaerobic digestion plant fed with pig slurry and stored for a long term in a lagoon.

This study combined different approaches to characterize organic sediments produced by an anaerobic digestion plant feed with pig slurry, and accumulated for many years in a lagoon. The results of all analyses identified a certain homogeneity of the sediments. As a consequence of the pig diet, the sediment contained an high concentration of Zn (about 4gkg-1) and Cu (about 1.2gkg-1), which were mostly associated to the particles with a size ranging from 2 to 53µm. The sediment was made of large amount of organic matter, mostly cellulose and recalcitrant molecules, and 30-40% mineral fraction. XANES and XES spectroscopies indicated the presence of zinc phosphate (38%), zinc sulfide (32%), zinc carbonate (19%), and zinc oxide (11%). The presence in the sediment of forms characterized by a very scarce solubility, as also confirmed by the Zn and Cu chemical speciation, indicated a low bioavailability of these metals. However, although their low mobility, the high concentrations of Zn and Cu allowed to consider the sediment not suitable to use as a fertiliser due to the potential risk of metal interaction with the food chain.

Solid anaerobic digestion batch of bio-waste as pre-treatment for improving amendment quality: The effect of inoculum recirculation.

The effect of solid anaerobic digestion batch (SADB) on bio-waste performed with and without inoculum on the quality of the final amendment was investigated by means of determining the content of organic carbon, humic and fulvic acids and the degree of humification. Two different processes were compared: composting and SADB with post-composting. Six parallel tests were performed. In three of these tests the SADB was inoculated mixing the bio-waste with the digestate from the previous run in a 1:1 ratio by weight. The amendment obtained by the SADB with post-composting treatment, in which the SADB was not inoculated, had an organic carbon content ranging from 15.5% TS to 30.3% TS resulting from 1% up to 14% higher than that of the corresponding composting processes. Similar results were achieved for the degree of humification. On the other hand SADB in which the inoculum was used generated about 300NL/kgVS of biogas instead of about 267NL/kgVS for non-inoculated runs.

Resveratrol ameliorates the maturation process of ß-cell-like cells obtained from an optimized differentiation protocol of human embryonic stem cells.

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic ß-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating ß-cell-like cells and demonstrates that RSV improves the maturation process.

Chemical and spectroscopic characterization of organic matter during the anaerobic digestion and successive composting of pig slurry.

In this work, anaerobic digestion of pig slurry and successive composting of the digestate after centrifugation were studied by means of chemical analysis, FTIR and fluorescence spectroscopy as excitation-emission matrix (EEM). Chemical analysis highlighted the organic matter transformation occurring during the processes. A decrease of volatile solids and total organic carbon were observed in the digestate with respect to the fresh pig slurry as a consequence of the consumption of sugars, proteins, amino acids and fatty acids used by microorganisms as a C source. Water Extractable Organic Matter (WEOM) was obtained for all samples and fractionated into a hydrophilic and a hydrophobic fraction. The highest WEOM value was found in the pig slurry indicating a high content of labile organic C. The digestate centrifuged and the digestate composted showed lower hydrophilic and higher hydrophobic contents because of the decrease of labile C. Total phenolic content was lower in the digestate with respect to fresh pig slurry sample (36.7%) as a consequence of phenolic compounds degradation. The strong decrease of total reducing sugars in the digestate (76.6%) as compared to pig slurry confirmed that anaerobic process proceed mainly through consumption of sugars which represent a readily available energy source for microbial activity. FTIR spectra of pig slurry showed bands indicative of proteins and carbohydrates. A drop of aliphatic structures and a decrease of polysaccharides was observed after the anaerobic process along with the increase of the peak in the aromatic region. The composted substrate showed an increase of aromatic and a relative decrease of polysaccharides. EEM spectra provided tryptophan:fulvic-like fluorescence ratios which increased from fresh substrate to digestate because of the OM decompostion. Composted substrate presented the lowest ratio due to the humification process.

Functional vascular smooth muscle-like cells derived from adult mouse uterine mesothelial cells.

In mammalian visceral organs, vascular smooth muscle cells (VSMCs) originate from an epithelial-to-mesenchymal transition (EMT) of embryonic mesothelial cells (MCs). The ability of adult MCs to recapitulate EMT and to acquire smooth muscle (SM) markers upon provasculogenic culture suggested they might retain embryonic vasculogenic differentiation potential. However, it remains unknown whether adult MCs-derived SM-like cells may acquire specific vascular SM lineage markers and the functionality of differentiated contractile VSMCs. Here, we describe how a gentle trypsinization of adult mouse uterine cords could selectively detach their outermost uterine mesothelial layer cells. As other MCs; uterine MCs (UtMCs) uniformly expressed the epithelial markers ß-catenin, ZO-1, E-cadherin, CD54, CD29, and CK18. When cultured in a modified SM differentiation media (SMDM) UtMCs initiated a loss of epithelial characteristics and gained markers expression of EMT (Twist, Snail, and Slug), stem and progenitor (Nanog, Sox2, C-kit, Gata-4, Isl-1, and nestin), SM (α-SMA, calponin, caldesmon, SM22α, desmin, SM-MHC, and smoothelin-B) and cardiac (BMP2, BMP4, ACTC1, sACTN, cTnI, cTnT, ANF, Cx43, and MLC2a). UtMCs repeatedly subcultured in SMDM acquired differentiated VSM-like characteristics and expressed smoothelin-B in the typical stress-fiber pattern expression of contractile VSMCs. Relevantly, UtMCs-derived VSM-like cells could generate "mechanical force" to compact collagen lattices and displayed in diverse degree voltage (K(+)) and receptor (endothelin-1, oxytocin, norepinephrine, carbachol and vasopressin)-induced [Ca(2+)](i) rises and contraction. Thus, we show for the first time that UtMCs could recapitulate in vitro differentiative events of early cardiovascular differentiation and transdifferentiate in cells exhibiting molecular and functional characteristics of VSMCs.

Greenhouse gas (GHG) emissions from soils amended with digestate derived from anaerobic treatment of food waste.

RATIONALE: The application of organic materials to agricultural lands is considered good practice to improve soil organic matter content and recycle nutrients for crop growth. The anaerobic treatment of food waste may have environmental benefits, particularly with regard to greenhouse gases (GHGs) mitigation and enhancement of carbon sequestration. METHODS: This work presents the results from a field experiment to evaluate CO(2) , CH(4) and N(2) O emissions from grassland amended with digestate produced by anaerobic fermentation of food waste. Experimental plots, located close to Rothamsted Research-North Wyke, were established using a randomized block design with three replicates and two treatments, added digestate (DG) and the unamended control (CNT). The digestate was applied on three occasions at an equivalent rate of 80 kg N ha(-1) . RESULTS: The application of digestate led to an increase in CO(2) emissions, especially after the 2(nd) application (74.1 kg CO(2) -C ha(-1) day(-1) ) compared with the CNT soil (36.4 kg CO(2) -C ha(-1) day(-1) ), whereas DG treatment did not affect the overall CH(4) and N(2) O emissions. The total grass yield harvested on a dry matter basis was greater in the DG treated plots (0.565 kg m(-2) ) than in the CNT plots (0.282 kg m(-2) ), as was the (15) N content in the harvest collected from the DG plots. CONCLUSIONS: The results suggest that the digestate can be applied to agricultural land as a fertilizer to grow crops. Our study was conducted in an exceptionally dry growing season, so conclusions about the effect of digestate on GHG emissions should take this into account, and further field trials conducted under more typical growing seasons are needed.