Ecotoxicological assessment of waste-derived organic fertilizers and long-term monitoring of fertilized soils using a multi-matrix and multi-species approach.

The present study investigates the ecotoxicity of 7 biofertilizers, including biowaste-derived organic matrices. Real-field tests were conducted to assess the impacts of soil fertilization with sewage sludge digestate from high-solid thermophilic anaerobic digestion (HSTAD) compared to those obtained on non-amended and urea-fertilized soils. The physical-chemical and ecotoxic impact of HSTAD digestate on soil was monitored for 12 months, at 5 time points and 2 soil depths, on a maize field divided in 3 portions (non-treated, fertilized with urea, amended with digestate). The chemical and physical characteristics of the soil were previously analyzed for 3 years to provide a long-term outlook of the impacts of biofertilizer application. Seven bioindicators were utilized for direct (on whole soil) and indirect (on soil elutriates) ecotoxicological tests on fertilizers and amended soils, including plant seeds (Lepidium sativum, Sorghum saccharatum, and Sinapsis alba), the aquatic organism Daphnia magna, the alga Raphidocelis subcapitata, the luminescent bacterium Aliivibrio fischeri, and the Nematode Caenorhabditis elegans. No serious negative effects on soil fertilized with HSTAD digestate were evidenced. Conversely, bioassays rather showed positive effects, encouraging the utilization of HSTAD digestate in agriculture, considering the proper concentrations of use. The obtained data were interpolated and a test battery integrated index was generated, confirming the absence of ecotoxicological risk for the soils amended with the applied fertilizers. The long-term evolution of the physical-chemical soil characteristics (including the concentrations of potential contaminants) was similar for both HSTAD digestate and urea application as well as for non-fertilized soil, indicating no negative effects due to digestate application on land. On the contrary, digestate application improved the content of stabilized organic matter and nutrients in soil. This study proposes a more correct approach to ecotoxicity assessment of fertilized soils for biofertilizer evaluation and demonstrates the long-term safe application of HSTAD digestate on agricultural soil.

Effects of the application of microbiologically activated bio-based fertilizers derived from manures on tomato plants and their rhizospheric communities.

Bio-based fertilizers (BBFs) recovered from animal manure are promising products to optimise resources recovery and generate high agricultural yields. However, their fertilization value may be limited and it is necessary to enrich BBFs with microbial consortia to enhance their fertilization value. Three specific microbial consortia were developed according to the characteristics of three different BBFs produced from manure (bio-dried solid fraction, solid fraction of digestate and biochar) to enhance plant growth and product quality. A greenhouse pot experiment was carried out with tomato plants grown with microbiologically activated BBFs applied either as N-organic fertilizers or as an organic amendment. A next generation sequencing analysis was used to characterise the development of each rhizospheric community. All the activated BBFs gave enhanced tomato yields (fresh and dry weight) compared with the non-activated treatments and similar to, or higher than, chemical fertilization. Concerning the tomato fruits' organoleptic quality, lycopene and carotenoids concentrations were improved by biological activation. Metagenomic analysis points at Trichoderma as the main driver of the positive effects, with the effects of added bacteria being negligible or limited at the early stages after fertilization. In the context of the circular economy, the activated BBFs could be used to replace synthetic fertilisers, reducing costs and environmental burdens and increasing production.

Microbial community acclimatization enhances bioplastics biodegradation and biogas production under thermophilic anaerobic digestion.

This paper reports the results of a novel study of microbial acclimatization for bioplastics anaerobic degradation and conversion into biogas. Three sequential anaerobic digestion (AD) runs were carried out to favour microbial acclimatization to two different bioplastics, starch-based (SBS) and polyactic-acid (PLA). AD of SBS and PLA bioplastics was favoured by the acclimatization of the inoculum to the substrate after each run of AD. SBS conversion into biogas increased by 52 % (from 94 to 143 NL kgVS-1) and it was correlated with the enhanced growth of starch degrading bacteria such as Hydrogenispora, Halocella and Haloplasma. PLA anaerobic degradation increased by 97 % (from 395 to 779 NLbiogas kgVS-1) and it was related to the acclimatization of known PLA-degraders such as Tepidimicrobium, Methanothermobacter and Tepidanaerobacter. Microbial acclimatization appears a suitable and low-cost strategy to enhance bioplastics circularity by promoting their anaerobic biodegradation and conversion into biogas.

Measuring the environmental impacts of sewage sludge use in agriculture in comparison with the incineration alternative.

This study compares two scenarios for sewage sludge treatment i.e., agricultural-land application (LA) and incineration (INC), in an Italian context (Pavia province, Po Valley). The study was realised within a regional project aiming to obtain useful data to better address future sludge management policies. To do so, an attributional Life Cycle Assessment (LCA) approach was chosen and the multi-functionality was addressed by using system expansion. Results indicated that the scenario INC had higher impacts than scenario LA for the categories linked to process inputs and to the direct emissions of incineration, such as Global warming potential (= + 60 %)., Stratospheric Ozone Depletion, Ozone Formation, Mineral Resource Scarcity and Fossil Resource Scarcity. System expansion i.e., the production of non-renewable fertilisers, played a large role (higher impacts) in the categories related to resource scarcity in the INC scenario. On the other hand, LA scenario showed higher impacts than INC for direct emissions due to fertilisation (Marine and Freshwater Eutrophication, and Particulate Matter). In conclusion, the use of sewage sludge in agriculture seemed to be competitive with the alternative of incineration but both sludge quality and emission reduction during sludge distribution in the field play an important role in the reduction of environmental impacts.

Influence of feedstock source on the development of polyhydroxyalkanoates-producing mixed microbial cultures in continuously stirred tank reactors.

Polyhydroxyalkanoates (PHAs) are the new frontier of bioplastic production; however, research is needed to develop and characterise efficient mixed microbial communities (MMCs) for their application with a multi-feedstock approach. Here, the performance and composition of six MMCs developed from the same inoculum on different feedstocks were investigated through Illumina sequencing to understand community development and identify possible redundancies in terms of genera and PHA metabolism. High PHA production efficiencies (>80% mg CODPHA mg-1 CODOA-consumed) were seen across all samples, but differences in the organic acids (OAs) composition led to different ratios of the monomers poly(3-hydroxybutyrate) (3HB) to poly(3-hydroxyvalerate) (3HV). Communities differed across all feedstocks, with enrichments in specific PHA-producing genera, but analysis of potential enzymatic activity identified a certain degree of functional redundancy, possibly leading to the general high efficiency seen in PHA production from all feedstocks. Leading PHAs producers across all feedstocks were identified in genera such as Thauera, Leadbetterella, Neomegalonema and Amaricoccus.

Pig Slurry Management Producing N Mineral Concentrates: A Full-Scale Case Study.

Manure treatment to recover nutrients presents a great challenge to delocalize nutrients from overloaded areas to those needing such nutrients. To do this, approaches for the treatment of manure have been proposed, and currently, they are mostly under investigation before being upgraded to full scale. There are very few fully operating plants recovering nutrients and, therefore, very few data on which to base environmental and economic studies. In this work, a treatment plant carrying out full-scale membrane technology to treat manure to reduce its total volume and produce a nutrient-rich fraction, i.e., the concentrate, was studied. The concentrate fraction allowed the recovery of 46% of total N and 43% of total P. The high mineral N content, i.e., N-NH4/total-N > 91%, allowed matching the REcovered Nitrogen from manURE (RENURE) criteria proposed by the European Commission to allow the potential substitution of synthetic chemical fertilizers in vulnerable areas characterized by nutrient overloading. Life cycle assessment (LCA) performed by using full-scale data indicated that nutrient recovery by the process studied, when compared with the production of synthetic mineral fertilizers, had a lower impact for the 12 categories studied. LCA also suggested precautions which might reduce environmental impacts even more, i.e., covering the slurry to reduce NH3, N2O, and CH4 emissions and reducing energy consumption by promoting renewable production. The system studied presented a total cost of 4.3 € tons-1 of slurry treated, which is relatively low compared to other similar technologies.

Impact of photobioreactor design on microalgae-bacteria communities grown on wastewater: Differences between thin-layer cascade and thin-layer raceway ponds.

Thin-layer (TL) photobioreactors (PBRs) are characterised by high productivity. However, their use is limited to lab/pilot-scale, and a deeper level of characterisation is needed to reach industrial scale and test the resistance of multiple microalgae. Here, the performance and composition of eight microalgal communities cultivated in the two main TLs design (thin-layer cascade (TLC) and thin-layer raceway pond (RW)) were investigated through Illumina sequencing. Chlorella vulgaris showed robustness in both designs and often acted as an "invasive" species. Inoculum and reactor type brought variability. Eukaryotic microalgae inocula led to a more robust and stable community (higher similarity), however, RWs were characterised by a higher variability and did not favour the eukaryotic microalgae. The only cyanobacterial inoculum, Nostoc piscinale, was maintained, however the community was variable between designs. The reactor design had an effect on the N cycle with the TLC and RW configurations, enhancing nitrification and denitrification respectively.

Nitrogen dynamics in soils fertilized with digestate and mineral fertilizers: A full field approach.

Highly stabilized digestate from sewage sludge and digestate-derived ammonium sulphate (RFs), were used in a comparison with synthetic mineral fertilizers (SF) to crop maize in a three-year plot trial in open fields. RFs and SF were dosed to ensure the same amount of mineral N (ammonia-N). In doing so, plots fertilized with digestate received much more N (+185 kg ha-1 of organic N) because digestate also contained organic N. The fate of nitrogen was studied by measuring mineral and organic N in soil at different depths, ammonia and N2O emissions, and N uptake in crops. Soil analyses indicated that at one-meter depth there was no significant difference in nitrate content between RF, SF and Unfertilized plots during crop season indicating that more N dosed with digestate did not lead to extra nitrate leaching. Ammonia emissions and N content in plants and grains measured were also similar for both RF and SF. Measuring denitrification activity by using gene makers resulted in a higher denitrification activity for RF than SF. Nevertheless, N2O measurements showed that SF emitted more N2O than RF (although it was not statistically different) (7.59 ± 3.2 kgN ha-1 for RF and 10.3 ± 6.8 kgN ha-1 for SF), suggesting that probably the addition of organic matter with digestate to RF, increased the denitrification efficiency so that N2 production was favoured. Soil analyses, although were not able detecting N differences between SF and Rf after three years of cropping, revealed a statistical increasing of total carbon, suggesting that dosing digestate lead to carbon (and maybe N) accumulation in soil. Data seem to suggest that N2O/N2 emission and organic N accumulation in soil can explain the fate of the extra N dosed (organic-N) in RF plots.

Environmental micro-niche filtering shapes bacterial pioneer communities during primary colonization of a Himalayas' glacier forefield.

The pedogenesis from the mineral substrate released upon glacier melting has been explained with the succession of consortia of pioneer microorganisms, whose structure and functionality are determined by the environmental conditions developing in the moraine. However, the microbiome variability that can be expected in the environmentally heterogeneous niches occurring in a moraine at a given successional stage is poorly investigated. In a 50 m2 area in the forefield of the Lobuche glacier (Himalayas, 5050 m above sea level), we studied six sites of primary colonization presenting different topographical features (orientation, elevation and slope) and harbouring greyish/dark biological soil crusts (BSCs). The spatial vicinity of the sites opposed to their topographical differences, allowed us to examine the effect of environmental conditions independently from the time of deglaciation. The bacterial microbiome diversity and their co-occurrence network, the bacterial metabolisms predicted from 16S rRNA gene high-throughput sequencing, and the microbiome intact polar lipids were investigated in the BSCs and the underlying sediment deep layers (DLs). Different bacterial microbiomes inhabited the BSCs and the DLs, and their composition varied among sites, indicating a niche-specific role of the micro-environmental conditions in the bacterial communities' assembly. In the heterogeneous sediments of glacier moraines, physico-chemical and micro-climatic variations at the site-spatial scale are crucial in shaping the microbiome microvariability and structuring the pioneer bacterial communities during pedogenesis.

Centrate as a sustainable growth medium: Impact on microalgal inocula and bacterial communities in tubular photobioreactor cultivation systems.

Centrate is a low-cost alternative to synthetic fertilizers for microalgal cultivation, reducing environmental burdens and remediation costs. Adapted microalgae need to be selected and characterised to maximise biomass production and depuration efficiency. Here, the performance and composition of six microalgal communities cultivated both on synthetic media and centrate within semi-open tubular photobioreactors were investigated through Illumina sequencing. Biomass grown on centrate, exposed to a high concentration of ammonium, showed a higher quantity of nitrogen (5.6% dry weight) than the biomass grown on the synthetic media nitrate (3.9% dry weight). Eukaryotic inocula were replaced by other microalgae while cyanobacterial inocula were maintained. Communities were generally similar for the same inoculum between media, however, inoculation with cyanobacteria led to variability within the eukaryotic community. Where communities differed, centrate resulted in a higher richness and diversity. The higher nitrogen of centrate possibly led to higher abundance of genes coding for N metabolism enzymes.

Profiling microalgal cultures growing on municipal wastewater and fertilizer media in raceway photobioreactors.

Microalgae cultivation is proposed as an effective system for pathogens reduction and wastewater depuration, however, a full characterisation of the risks is still needed. Two raceways were inoculated with Scenedesmus, one using wastewater and the other using a fertilizer medium. Microbial community and pathogen presence were explored by next generation sequencing (NGS), commercial qPCR array and plate counts. These methods proved to be complementary for a full characterization of community structure and potential risks. Media and sampling locations contributed to shape communities and pathogenic loads. The main pathogenic genera detected were Arcobacter and Elizabethkingia (mainly in wastewater) with an important presence of Aeromonas (all samples). A lower presence of pathogens was detected in fertilizer samples, while wastewater showed a reduction from inlet to outlet. Raceways showed potential as an effective biotreatment, with most of the retained pathogens released in the outlet and only a minor part settled in the biomass.

Life cycle assessment of Parmigiano Reggiano PDO cheese with product environmental footprint method: A case study implementing improved slurry management strategies.

The environmental impact of Parmigiano Reggiano PDO cheese was quantified using the Product Environmental Footprint Category Rules (PEFCRs) in a Traditional System (TS) and in an Improved Management System (IMS). The TS differs from IMS with respect to slurry management (raw slurry storage vs anaerobic digestion and storage of the liquid fraction of digestate) and application of nutrients to the field (by slurry tanker with a diverter plate vs soil injection at pre-sowing and side dressing). Two additional scenarios were evaluated by considering the possible environmental enhancement achievable by reducing enteric methane production and by using soybean grain produced in Italy as the protein source for animals' diets. The environmental impact was quantified both for 1 kg of fat and protein corrected milk (FPCM) and for the production of 10 g dry matter equivalent of cheese as single score. For the first assessment, the environmental impact results were 124 and 112 µPt kg FPCM-1 for TS and IMS, respectively. In the second case, it was 10.8 µPt and 9.9 µPt 10 g dry matter equivalent-1 of cheese, for TS and IMS, respectively. The specific cost for reducing the GHG emissions in this production chain was equal to 34 € Mg-1 milk produced. Finally, although specific studies should consider the reduction of enteric methane emissions and the use of soybean grain nationally produced as feed source, the scenarios evaluated in this study highlighted some potential for environmental improvements. Even small environmental improvements to the Parmigiano Reggiano PDO cheese supply chain can bring substantial improvements to the sustainability of the food market, because of the widespread demand on the global market of Parmigiano Reggiano and of its chance of attracting consumers who are sensitive to environmental problems.

Assessing the anaerobic degradability and the potential recovery of biomethane from different biodegradable bioplastics in a full-scale approach.

The aim of the present study was to evaluate the anaerobic degradability and the potential recovery of biomethane from different bioplastics using a full-scale approach. Bioplastics were placed inside a real anaerobic digestion plant working under thermophilic conditions and quantitative and qualitative degradation of bioplastics was evaluated. Laboratory-scale experiments were used to determine the amount of biomethane produced by anaerobic degradation of bioplastics. Polylactic acid-based items may degrade completely using retention times compatible with anaerobic digestion plants contributing positively to biomethane production, i.e., in 90 days 397 ± 8 NL CH4 kgvolatile solids-1 were produced by polylactic acid-based cutlery. Starch-based shoppers showed a quick degradation of the starch component in the first month of anaerobic digestion, followed by a slow degradation of the polyester component. Anaerobic digestion and/or anaerobic digestion coupled to digestate composting may represent the best strategy to dispose these wastes meeting the principles of Circular Economy.

Brachytic2 mutation is able to counteract the main pleiotropic effects of brown midrib3 mutant in maize.

Maize is the basis of nutrition of domesticated herbivores and one of the most promising energy crops. The presence of lignin in the cell wall, tightly associated to carbohydrates, prevents the physical access of enzymes such as cellulase, limiting the carbohydrate degradability and consequently the energy value. To increase the utilization of the biomass cellulose content, the challenge of breeding programs is to lower or modify the lignin components. In maize several mutations are able to modify the lignin content and in particular the mutation in brown midrib3 (bm3) gene appeared as one of the most promising in breeding programs. Unfortunately this mutation has several negative pleiotropic effects on various important agronomic traits such as stay green, lodging and susceptibility to several infections.The maize Brachyitic 2 (br2) gene encodes for a putative protein involved in polar movement of auxins. br2 mutant plants are characterized by shortening of lower stalk internodes, unusual stalk strength and tolerance to wind lodging, darker leaves persisting longer in the active green state in comparison to wild type plants, suggesting a possible utilization of br2 plants to counteract the negative effects of the bm3 mutation. In this work, we report the generation and a preliminary characterization of the double mutant bm3 br2, suggesting the potential use of this new genetic material to increase biomass cellulose utilization.

Growth Performance, Biochemical Composition and Nutrient Recovery Ability of Twelve Microalgae Consortia Isolated from Various Local Organic Wastes Grown on Nano-Filtered Pig Slurry.

This paper demonstrated the growth ability of twelve algae-microbial consortia (AC) isolated from organic wastes when a pig slurry-derived wastewater (NFP) was used as growth substrate in autotrophic cultivation. Nutrient recovery, biochemical composition, fatty acid and amino acid profiles of algae consortia were evaluated and compared. Three algae-microbial consortia, i.e., a Chlorella-dominated consortium (AC_1), a Tetradesmus and Synechocystis co-dominated consortium (AC_10), and a Chlorella and Tetradesmus co-dominated consortium (AC_12) were found to have the best growth rates (µ of 0.55 ± 0.04, 0.52 ± 0.06, and 0.58 ± 0.03 d-1, respectively), which made them good candidates for further applications. The ACs showed high carbohydrates and lipid contents but low contents of both proteins and essential amino acids, probably because of the low N concentration of NFP. AC_1 and AC_12 showed optimal ω6:ω3 ratios of 3.1 and 3.6, which make them interesting from a nutritional point of view.

Using highly stabilized digestate and digestate-derived ammonium sulphate to replace synthetic fertilizers: The effects on soil, environment, and crop production.

Recovered fertilizers (a highly stabilized digestate and ammonium sulphate) obtained from anaerobic digestion of sewage sludge, were used on plot trials with a maize crop, in a comparison with synthetic fertilizers. After three consecutive cropping seasons, the soils fertilized with the recovered fertilizers (RF), compared to those fertilized with synthetic fertilizers (SF), did not show significant differences either in their chemical characteristics or in the accumulation of inorganic and organic pollutants (POPs). The RF ensured an ammonia N availability in the soil equal to that of the soil fertilized with SF, during the whole period of the experiment. Furthermore, no risks of N leaching were detected, and the use of RF did not result in a greater emission of ammonia or greenhouse gases than the use of SF. The agronomic results obtained using RF were equivalent to those obtained with SF (fertilizer use efficiency of 85.3 ± 10 and 93.6 ± 4.4% for RF and SF respectively). The data show that utilising a very stable digestate can be a good strategy to produce a bio-based fertilizer with similar performance to that of a synthetic fertilizer, without environmental risks.

Environmental Performance in the Production and Use of Recovered Fertilizers from Organic Wastes Treated by Anaerobic Digestion vs Synthetic Mineral Fertilizers.

Recovered fertilizers (RFs), in the form of digestate and digestate-derived ammonium sulfate, were produced from organic wastes by thermophilic anaerobic digestion (AD) at full scale. RFs were then used for crop production (maize), substituting synthetic mineral fertilizers (SFs). Environmental impacts due to both RF and SF production and use were studied by a life cycle assessment (LCA) approach using, as much as possible, data directly measured at full scale. The functional unit chosen was referred to as the fertilization of 1 ha of maize, as this paper intends to investigate the impacts of the use of RF (Scenario RF) for crop fertilization compared to that of SF (Scenario SF). Scenario RF showed better environmental performances than the system encompassing the production and use of urea and synthetic fertilizers (Scenario SF). In particular, for the Scenario RF, 11 of the 18 categories showed a lower impact than the Scenario SF, and 3 of the categories (ionizing radiation, fossil resource scarcity, and water consumption) showed net negative impacts in Scenario RF, getting the benefits from the credit for renewable energy production by AD. The LCA approach also allowed proposing precautions able to reduce further fertilizer impacts, resulting in total negative impacts in using RF for crop production. Anaerobic digestion represents the key to propose a sustainable approach in producing renewable fertilizers, thanks to both energy production and the modification that occurs to waste during a biological process, leaving a substrate (digestate) with high amending and fertilizing properties.

Diversifying the products from the organic fraction of municipal solid waste (OFMSW) by producing polyhydroxyalkanoates from the liquid fraction and biomethane from the residual solid fraction.

This study describes the diversification of products obtainable from the organic fraction of municipal solid waste (OFMSW) by producing polyhydroxyalkanoates (PHA) from the liquid fraction and biomethane from the residual solid fraction. OFMSW samples were taken during the 2021 season from two full field scale plants treating wastes. After solid/liquid (S/L) separation, 80% of initial organic acids (OAs) were released in the liquid stream. OAs were then used as feed for PHA production and residual solid cakes were tested for biomethane production. Complete mass balance and energy balance were calculated. PHAs production was of 115 ± 23 (n = 6) g kg- 1 OFMSW (TS) and residual biomethane of 219 ± 3 g kg- 1 OFMSW TS, (n = 6). Energy balance indicated that nearly 40% of OFMSW energy was recovered as products. This value was lower than that obtained previously when AD was performed before OAs separation (i.e. 64%).

The structure and diversity of microalgae-microbial consortia isolated from various local organic wastes.

Pure microalgae cultivation in organic wastes may be hampered by their low adaptation to extreme growth conditions and by the risk of microbial contamination. This work aimed to isolate self-adapted microalgae-microbial consortia able to survive in organic wastes characterized by extreme conditions, to be then proposed for technological application in removing carbon and nutrients from wastes' streams. To do so, sixteen organic wastes with different origins and consistency were sampled. Twelve microbial consortia were isolated from wastes and their eukaryotic and prokaryotic compositions were analyzed by next generation sequencing. Eight eukaryotic communities were dominated by Chlorophyta, led by Chlorella, able to survive in different wastes regardless of chemical-biological properties. Tetradesmus, the second most represented genus, grew preferentially in substrates with less stressing chemical-physical parameters. Chlorella and Tetradesmus were mostly isolated from cow slurry and derived wastes which proved to be the best local residual organic source.

Polyphenol bioactivity evolution during the spontaneous fermentation of vegetal by-products.

Food industry by-products such as grape pomace (GP), tomato pomace (TP), and spent coffee grounds (SCG) are rich in polyphenols (PP) but are easily biodegradable. The aim of this study is to test Spontaneous Fermentation (SF) as treatment to modify PP profile and bioactivity. The results highlighted that the by-products' organic matter and the microbial populations drove the SF evolution; heterolactic, alcoholic, and their mixtures were the predominant metabolisms of TP, GP, and SCG + GP co-fermentation. Increases in the extractable amounts and antiradical activity occurred for all the biomasses. Regarding the aglycate-PPs (APP), i.e. the most bioreactive PPs, significant changes occurred for TP and GP but did not influence the anti-inflammatory bioactivity. The co-fermentation increased significantly chlorogenic acid and consumed most of the APPs, acting as a purification system to obtain a highly concentrated APP fraction, so that the extract might be employed for a specific purpose.