Development of functional consortia for the pretreatment of compostable lignocellulosic waste: A simple and effective solution to a large-scale problem.

Microorganisms drive the degradation of organic matter thanks to their enzymatic versatility. However, the structure of lignocellulose poses a great challenge for the microbiota inhabiting a compost pile. Our purpose was to increase the biodegradability of vegetable waste in the early stages of the composting process by applying a microbial consortium with lignocelllulolytic capacity. For this, a previous screening was performed among the culturable microbiota from different composting processes to find inoculants with ligninocellulolytic activity. Selected strains were applied as a pure culture and as a microbial consortium. The starting material was composed of tomato plant and pruning remains mixed in a ratio (50:50 v/v), whose humidity was adjusted to around 65%. To determine the ability of both treatments to activate the biodegradation of the mixtures, moisture, organic matter, ash, C/N ratio, 4-day cumulative respirometric index (AT4) and degradation rates of cellulose, hemicellulose and lignin were evaluated. Subsequently, a real composting process was developed in which the performance of the microbial consortium was compared with the composting process without inoculum (control). According to our tests, three microbial strains (Bacillus safensis, Bacillus licheniformis and Fusarium oxysporum) were selected. The results showed that the application of the bacteria strains at low doses (104 CFU g-1 on the complete residual material of the pile) resulted in higher rates of lignocelullose degradation after 10 days of treatment compared to that observed after application of the fungus in pure culture or untreated controls. The implementation of the strategy described in this work resulted in obtaining compost with better agronomic quality than the uninoculated controls. Therefore, the application of this consortium could be considered as an interesting tool for bioactivation of lignocellulosic waste prior to the composting process.

Sustainable approach to the control of airborne phytopathogenic fungi by application of compost extracts.

The search for new sustainable alternatives for plant disease control has gained interest in the last decades. Compost extracts are nowadays considered a remarkable alternative to agrochemicals due to their biopesticidal properties. However, these properties could be affected by the different variables of extraction protocols and by starting compost. This work focused on the physicochemical and biological characterization of compost extracts obtained from a wide range of composted materials and different extraction protocols (CEP). CEP-1 and CEP-4 involved incubation at 20 °C for 48 h and 14 days, respectively; CEP-2 incubation for 24 h at 40 °C; while CEP-3 were incubated for 12 h at 70 °C. Electrical conductivity, pH, total organic carbon (TOC) and phenolic content were determined as well as the actinobacterial count and enzyme profiles related to plant pathogen suppression. Additionally, the influence of the different materials and protocols on the in vitro growth inhibition of Alternaria alternata and Botrytis cinerea was determined. The starting materials and extraction protocols significantly influenced the physicochemical and biological characteristics of extracts. Treatments based on long incubation times at 20 °C, as well as those based on short incubation times at 40 °C, resulted in extracts with increased suppressive properties. However, extracts derived from CEP-3 protocol were characterized by high phenolic and TOC content, low functional biodiversity, and a more discreet antagonistic capacity. Therefore, the development and optimization of suitable extraction protocols could lead to compost extracts with increased phytoprotective capacities, thus becoming an effective and sustainable alternative to chemical inputs.

Effect of agricultural microplastic and mesoplastic in the vermicomposting process: Response of Eisenia fetida and quality of the vermicomposts obtained.

This work evaluates the effect of agricultural plastic waste (APW) in two particle sizes, microplastic and film debris, and subjected to a pre-treatment by exposure to UV-C, in the development of the vermicomposting process. Eisenia fetida health status and metabolic response and the vermicompost quality and enzymatic activity were determined. The environmental significant of this study is mainly related to how can affect plastic presence (depending on plastic type, size and/or if it is partially degraded) not only to this biological process of organic waste degradation, but also to the vermicompost characteristics, since these organic materials will be reintroduced in the environment as organic amendments and/or fertilizers in agriculture. The plastic presence induced a significant negative effect in survival and body weight of E. fetida with an average decrease of 10% and 15%, respectively, and differences on the characteristics of the vermicomposts obtained, mainly related with NPK content. Although the plastic proportion tested (1.25% f. w.) did not induce acute toxicity in worms, effects of oxidative stress were found. Thus, the exposure of E. fetida to AWP with smaller size or pre-treated with UV seemed to induce a biochemical response, but the mechanism of oxidative stress response did not seem to be dependent on the size or shape of plastic fragments or pre-treated plastic.

Mitigation of phytotoxic effect of compost by application of optimized aqueous extraction protocols.

The abuse of chemical fertilizers in recent decades has led the promotion of less harmful alternatives, such as compost or aqueous extracts obtained from it. Therefore, it is essential to develop liquid biofertilizers, which in addition of being stable and useful for fertigation and foliar application in intensive agriculture had a remarkable phytostimulant extracts. For this purpose, a collection of aqueous extracts was obtained by applying four different Compost Extraction Protocols (CEP1, CEP2, CEP3, CEP4) in terms of incubation time, temperature and agitation of compost samples from agri-food waste, olive mill waste, sewage sludge and vegetable waste. Subsequently, a physicochemical characterization of the obtained set was performed in which pH, electrical conductivity and Total Organic Carbon (TOC) were measured. In addition, a biological characterization was also carried out by calculating the Germination Index (GI) and determining the Biological Oxygen Demand (BOD5). Furthermore, functional diversity was studied using the Biolog EcoPlates technique. The results obtained confirmed the great heterogeneity of the selected raw materials. However, it was observed that the less aggressive treatments in terms of temperature and incubation time, such as CEP1 (48 h, room temperature (RT)) or CEP4 (14 days, RT), provided aqueous compost extracts with better phytostimulant characteristics than the starting composts. It was even possible to find a compost extraction protocol that maximize the beneficial effects of compost. This was the case of CEP1, which improved the GI and reduced the phytotoxicity in most of the raw materials analyzed. Therefore, the use of this type of liquid organic amendment could mitigate the phytotoxic effect of several composts being a good alternative to the use of chemical fertilizers.

Design and validation of cyanobacteria-rhizobacteria consortia for tomato seedlings growth promotion.

The use of rhizobacteria provide great benefits in terms of nitrogen supply, suppression of plant diseases, or production of vitamins and phytohormones that stimulate the plant growth. At the same time, cyanobacteria can photosynthesize, fix nitrogen, synthesize substances that stimulate rhizogenesis, plant aerial growth, or even suppose an extra supply of carbon usable by heterotrophic bacteria, as well as act as biological control agents, give them an enormous value as plant growth promoters. The present study focused on the in vitro establishment of consortia using heterotrophic bacteria and cyanobacteria and the determination of their effectiveness in the development of tomato seedlings. Microbial collection was composed of 3 cyanobacteria (SAB-M612 and SAB-B866 belonging to Nostocaceae Family) and GS (unidentified cyanobacterium) and two phosphate and potassium solubilizing heterotrophic bacteria (Pseudomonas putida-BIO175 and Pantoea cypripedii-BIO175). The results revealed the influence of the culture medium, incubation time and the microbial components of each consortium in determining their success as biofertilizers. In this work, the most compatible consortia were obtained by combining the SAB-B866 and GS cyanobacteria with either of the two heterotrophic bacteria. Cyanobacteria GS promoted the growth of both rhizobacteria in vitro (increasing logarithmic units when they grew together). While Cyanobacteria SAB-B866 together with both rhizobacteria stimulated the growth of tomato seedlings in planta, leading to greater aerial development of the treated seedlings. Parameters such as fresh weight and stem diameter stood out in the plants treated with the consortia (SAB-B866 and both bacteria) compared to the untreated plants, where the values doubled. However, the increase was more discrete for the parameters stem length and number of leaves. These results suggest that the artificial formulation of microbial consortia can have positive synergistic effects on plant growth, which is of enormous agro-biotechnological interest.

Biorecovery of olive mill wastewater sludge from evaporation ponds.

Olive mill wastewater (OMW) resulting from the olive oil extraction process is usually disposed of in evaporation ponds where it concentrates generating a sludge that pollutes the ponds nearby area. In this study, four bio-treatments were applied for the in-situ bioremediation and valorization of OMW sludge: Landfarming, phytoremediation, composting and vermicomposting. In all cases, the OMW sludge was added with organic residues (mushroom compost, rabbit manure, and chicken manure). The bio-treatments were carried out in duplicate, inoculated and non-inoculated, to determine the effect of a specialized fungal consortium (Aspergillus ochraceus H2 and Scedosporium apiospermum H16) on the efficacy of the bio-treatments. The evaluation of chemical parameters, toxicity, and functional microbial biodiversity revealed that the four techniques depleted the toxicity and favored the stimulation of functional microbiota. Landfarming and phytoremediation allowed the decontamination and improvement of soils. Composting and vermicomposting also offered high-quality products of agronomic interest. Inoculation improved the bioremediation effectiveness. Biological treatments are effective for the safe recovery of contaminated OMW sludge into high-quality services and products.

Seed biopriming with cyanobacterial extracts as an eco-friendly strategy to control damping off caused by Pythium ultimum in seedbeds.

This work highlights the ability of various cyanobacterial extracts from Anabaena spp., Tolypothrix spp., Nostoc or Trichormus, among others genera, to control the incidence of damping-off caused by Pythium ultimum in cucumber seedlings. Protocols applied aimed at the preliminary characterization of the cyanobacterial collection were very useful for predicting their phytotoxic, phytostimulating and biopesticidal capacity. First, the phytostimulatory or phytotoxic potential of a collection of 31 sonicated cyanobacterial extracts was analyzed by calculating the germination index in watercress seeds and the increase or loss of seedling weight. Likewise, the collection was characterized according to its ability to inhibit the growth of P. ultimum by dual culture bioassays and detached-leaf test. Finally, after selecting the most effective extracts, a preventive damping-off bioassay was performed based on cucumber seed biopriming. The strain SAB-M465 showed to be the most efficient strain against the in vitro growth of P. ultimum, while SAB-B912 was more discreet in this regard, but proved to be the most effective as a germination stimulator. Seed biopriming strategy with sonicated extracts of cyanobacteria revealed a remarkable promoter effect in the early stages of plant development, although only SAB-M465 was positioned as an effective control agent against damping-off caused by P. ultimum in cucumber seedbeds.

Revisiting the succession of microbial populations throughout composting: A matter of thermotolerance.

Composting has been traditionally considered a process in which a succession of mesophilic and thermophilic microbial populations occurs due to temperature changes. In order to deepen in this model, 1380 bacterial and fungal strains (the entire culturable microbiota isolated from a composting process) were investigated for their ability to grow across a wide range of temperatures (20 to 60 °C). First, qualitative tests were performed to establish a thermal profile for each strain. Then, quantitative tests allowed ascertaining the extent of growth for each strain at each of the tested temperatures. The identity of the isolates enabled to position them taxonomically and permitted tracking the strains throughout the process. Results showed that 90% of the isolates were classified as thermotolerant (they grew at all tested temperatures). Only 9% and 1% of the studied strains showed to be strictly mesophilic or thermophilic, respectively. Firmicutes exhibited the greatest thermal plasticity, followed by Actinobacteria and Ascomycota. Most of the Proteobacteria and all Basidiomycota strains were also able to grow at all the assayed temperatures. Thermotolerance was clearly demonstrated among the composting microbiota, suggesting that the idea of the succession of mesophilic and thermophilic populations throughout the process might need a reassessment.

Uncovering new indicators to predict stability, maturity and biodiversity of compost on an industrial scale.

Currently, the metagenomic study of the composting process has gained great importance since it has allowed the identification of the existence of microorganisms that, until now, had not been isolated during the process by traditional techniques. However, it is still complex to determine which bioindicators could reveal the degree of maturity and stability of a particular compost. Thereby, the main objective of this work was to demonstrate the possible correlation between traditional parameters of maturity and stability of compost, with other indicators of biodiversity in products highly heterogeneous from composting processes on an industrial scale. The results demonstrated the enormous influence of the raw materials in characterizing the products obtained. Even so, important relationships were established between the Chao1 and Shannon indexes, and certain parameters related to the maturity, stability and toxicity of the samples, such as nitrification index, humification rate, phenolic content, germination index or oxygen consumption.

Prospection of cyanobacteria producing bioactive substances and their application as potential phytostimulating agents.

This work clarifies some of the substances involved with the biostimulant effect shown by 28 cyanobacteria isolated from different aquatic environments. The production of salicylic acid, cytokinins, siderophores and phosphate solubilization were analyzed in vitro, as well as the phytostimulant/phytotoxic effect on watercress seeds at two different extract concentrations (0.5 and 0.2 mg mL-1). The most prominent plant growth promoting cyanobacteria were verified in vivo at two different doses (0.5 and 0.1 mg mL-1). 21.4 % and 7.1 % of the tested strains produced siderophores or phosphate solubilization, respectively. The production of salicylic acid was stood out for the strains Calothrix SAB-B797, Nostoc SAB-B1300 and Nostoc SAB-M612, while Nostoc SAB-M251 and Trichormus SAB-M304 were noticeable regard to cytokinin production. The highest values of germination occurred when the extracts were applied in low dose (0.5 mg mL-1). Nostoc SAB-M612 provoked the stimulation of aerial and radicular growth in cucumber seedlings.

Evaluating the influence of raw materials on the behavior of nitrogen fractions in composting processes on an industrial scale.

Success of composting as an ecological technology for organic waste management has allowed its implementation in the current circular economy models. However, composting on an industrial scale often shows drawbacks and peculiarities. In this work, a comparative analysis of 15 industrial composting facilities was carried out in which different anthropogenic organic waste were processed. Results showed that composting process on an industrial scale did not always evolve in a standard way. Monitoring parameters as well as enzymatic activity depended largely on the raw materials and were strongly linked to the transformation of nitrogen fractions. Despite the heterogeneity of the processes and raw materials, microbial activity managed to the optimal biotransformation, obtaining products that comply with the agronomic quality standards. This work represents a breakthrough in composting and provides new knowledge for better management of this process on an industrial scale.

Enzymatic profiles associated with the evolution of the lignocellulosic fraction during industrial-scale composting of anthropogenic waste: Comparative analysis.

In the new European Waste Law, composting is proposed as one of the best options to properly manage organic waste of anthropogenic origin. Currently, the massive generation of this type of waste, as well as its heterogeneity, makes difficult in many cases control this process of degradation on an industrial scale. In this work, 15 facilities were selected based on 5 types of organic waste: Urban Solid Waste, Vegetable Waste, Sewage Sludges, Agrifood Waste and "Alpeorujo". The samples were collected in different thermal phases. The results revealed very different physicochemical and enzymatic profiles, as well as different degrees of humification depending on the process and the raw materials. However, parameters such as ß-glucosidase, amylase, lignin/holocellulose ratio and humification rate showed similar trends in all cases. All of them could act as important indicators to evaluate the quality of a composting process, despite the heterogeneity of the starting materials.

Bioprospecting from plant waste composting: Actinobacteria against phytopathogens producing damping-off.

Strains isolated during composting processes of plant waste, and identified as Actinobacteria, proved to be significant producers of compounds that actively participate in the control of phytopathogens, such as those that cause Damping-off disease. Although most of the actinomycetes analyzed showed to be antagonistic strains against common phytopathogens, only some 30% proved to be capable of producing bioactive substances, such as siderophores, salicylic acid, chitinase enzymes or cyanide, so that antibiosis could be considered the most probable antagonistic mechanism for a high proportion of the strains investigated. 6% of the microorganisms identified in this work, were selected as potential strains to be investigated in depth, since they further stimulated plant growth (germination index tests greater than 100%). Microbacteriaceae was one of the most prominent families.

Enhanced turnover of organic matter fractions by microbial stimulation during lignocellulosic waste composting.

Enhanced organic matter turnover was detected in lignocellulosic composting piles inoculated with microorganisms specifically capable of decomposing polymeric compounds. In comparison to uninoculated piles, the following results were obtained in the inoculated piles: degradation of hemicellulose, cellulose and lignin were 28%, 21% and 25% respectively higher. Total organic matter, total sugars and phenolic compounds also decreased more intensely. Greater amounts of soluble organic carbon, reducing sugars and soluble proteins were available to the composting microbiota. Recycling of organic to inorganic nitrogen was improved and humification was more intense and earlier attained. Microbial community structure was also affected by inoculation. It was initially thought that these effects were due to enzymatic capabilities of inoculants, however, microbial counts, especially those corresponding to functional groups, revealed that inoculation induced a true stimulation of microbial growth and activity in the entire composting microbiota which was actually responsible for all the beneficial effects reported here.

Dynamics of bacterial microbiota during lignocellulosic waste composting: Studies upon its structure, functionality and biodiversity.

An intensive isolation program carried out in three replicated composting piles allowed the identification of the resident and transient components of the composting microbiome. More than 4000 bacterial strains were isolated, enzymatically characterized and identified by partial sequencing of their 16S rRNA gene. While microorganisms isolated under mesophilic conditions were prominent throughout the process, thermophilic stages gathered the highest total counts and spore-forming bacteria prevailed at the bio-oxidative phase of composting. Enzymatic capabilities related to the degradation of polymeric materials were exhibited by most of the isolates and as a result of these activities, more soluble compounds could be made available to the entire composting microbiota. A high proportion of isolates showed to be thermotolerant as they were detected at mesophilic and thermophilic phases. Isolated strains belonged to 187 bacterial species. Biodiversity was greater at the central stages of composting and mesophilic, thermophilic and cooling phases shared 50% of species.

Evolution of enzymatic activities and carbon fractions throughout composting of plant waste.

Many alternatives for the proper disposal of horticultural plant wastes have been studied, and composting is one of the most attractive due to its insignificant environmental impact and low cost. The quality of compost for agronomical use is related to the degree of organic matter maturation and stabilization. Traditional parameters as well as temperature, ratio C/N, cationic exchange capacity, extractable carbon, or evolution of humificated substances have been successfully used to assess compost maturity and stability. However, microorganisms frequently isolated during composting release a wide range of hydrolytic enzymes, whose activity could apparently give interesting information on the rate of decomposition of organic matter and, therefore, on the product stability. The aim of this work was to study the evolution of some important enzymatic activities during composting of agricultural wastes and their comparison with other chemical parameters commonly employed as quality and maturity indexes, to establish a relationship between the degradation intensity of specific organic carbon fractions throughout the process. In this work, the chemical and biochemical parameters of plant wastes were studied along a composting process of 189 days to evaluate their importance as tools for compost characterization. Results showed an intense enzymatic activity during the first 2-3 weeks of composting (bio-oxidative phase), because of the availability of easily decomposable organic compounds. From a biological point of view, a less intense phase was observed between second and third month of composting (mesophilic or cooling phase). Finally, chemical humification parameters were more closely associated with the period between 119 and 189 days (maturation phase). Significant correlations between the enzymatic activities as well as between enzyme activities and other more traditional parameters were also highlighted, indicating that both kind of indexes can be a reliable tool to determine the degree of stability and maturation of horticultural plant wastes based-compost.

Use of chemometrics in the chemical and microbiological characterization of composts from agroindustrial wastes.

Physico-chemical, chemical and microbiological parameters were studied in a set of fifteen different composts elaborated with agroindustrial wastes using two different composting systems (turning and static pile composting). To carry out the chemometric evaluation, multivariate statistical analysis techniques, such as hierarchical cluster analysis (HCA) and factorial analysis (FA) were used. Composts obtained showed suitable physico-chemical and chemical properties for their use as organic amendment and a good maturity degree. HCA allowed to classify the organic materials mainly in four groups: cluster A, cluster B, cluster C and unclustered composts; also, this statistical tool showed the lack of influence of the composting system in the final characteristics of these composts. On the other hand, through FA, it was possible to identify the principal variables associated to the composting of agroindustrial wastes in four factors that explained 72.3% of the variability.

Microbial population dynamics and enzyme activities in composting processes with different starting materials.

A biological comparison based on differences in the starting material for composting processes was made. Mesophilic aerobic microbiota, fungi, actinomycetes and hemicellulolytic microorganisms reached significantly higher levels in the MSW final product. The population of cellulolytic microorganisms did not show a clear trend, although it was more numerous in the HW piles. Counts for N(2)-fixing and ammonifying bacteria were significantly higher in the SS pile at the early stages of the process, while populations tended to become equal as time progressed. The lowest populations were detected for nitrifying bacteria, with higher but not always significant levels for the SS pile. beta-Glucosidase and phosphatase activities were higher in the SS pile at the early stages. Protease reached its maximum activity during the bio-oxidative phase and final stages in the HW and MSW piles, respectively. Dehydrogenase activity, with an occasional high level for the MSW at the beginning of the process, was almost inexistent since the end of the bio-oxidative phase. On the contrary, urease showed higher levels at the final stage of the process, with the MSW pile showing the greatest levels most of the time. According to these results, the nature of the starting material causes differences in biological parameters.

Evolution of the pathogen content during co-composting of winery and distillery wastes.

The aim of this study was to monitor some microbial indicators and pathogen contents (sulphite reducers clostridia, total enterobacteriaceae, total coliforms, faecal coliforms (Escherichia coli), enterococci, Staphylococcus aureus and Salmonella spp.) throughout the co-composting of wastes from the winery and distillery industry with other organic residues, as well as the effect of the composting system used. Seven different piles using mixtures of winery-distillery wastes with other organic materials were prepared. P1 and P2 were made using grape stalk (GS), grape marc (GM), exhausted grape marc (EGM) and sewage sludge (SS), whereas in P3 and P4 were also used exhausted grape marc with cow manure (CW) and poultry manure (PM), respectively, using the Rutgers system. Additionally, P2 was watered with vinasse (V). The rest of piles (P5, P6 and P7) were prepared with grape marc, exhausted grape marc, cow manure and poultry manure, using the turning system. The effectiveness of the composting process to reduce the pathogen content was higher in the static aerated piles than in those elaborated with the turning. The relatively high temperatures (50-60 degrees C) reached in some of the piles produced a notable decrease in some microbial groups, such as total and faecal coliforms (E. coli), but the characteristics of the raw materials used notably influenced the pathogen contents of the end-product.