Enhancing earthworm (Lumbricus terrestris) tolerance to plastic contamination through gut microbiome fortification with plastic-degrading microorganisms.

Microorganisms from L. terrestris gut previously exposed to different types of plastic (PET, LDPE, LLDPE, and PS) were studied to be used as probiotics of earthworms in plastic-contaminated soils (LDPE, LLDPE and recycled mulching film) at mesocosm-scale trials. The most abundant morphotypes with enzymatic capacities of interest were identified. Pseudomonas alkylphenolica (PL4) and Pseudomonas putida (PL5) strains were selected to be used as inoculants using Morus alba leaves as carriers to strengthen the intestinal microbiota of earthworms. Culture (selective cetrimide agar medium) and molecular (qPCR) techniques were used to trace the presence of the inoculum in the intestine of the earthworms. Additionally, a metataxonomic analysis was carried out to study the biodiversity and functionality of the earthworm microbiome, and their measure of survival and weight. Probiotics improved the survival rates of earthworms exposed to plastics, which also increased the abundance of microbial groups of interest in plastic bioremediation tasks.

Development of plastic-degrading microbial consortia by induced selection in microcosms.

The increase in the production of highly recalcitrant plastic materials, and their accumulation in ecosystems, generates the need to investigate new sustainable strategies to reduce this type of pollution. Based on recent works, the use of microbial consortia could contribute to improving plastic biodegradation performance. This work deals with the selection and characterization of plastic-degrading microbial consortia using a sequential and induced enrichment technique from artificially contaminated microcosms. The microcosm consisted of a soil sample in which LLDPE (linear low-density polyethylene) was buried. Consortia were obtained from the initial sample by sequential enrichment in a culture medium with LLDPE-type plastic material (in film or powder format) as the sole carbon source. Enrichment cultures were incubated for 105 days with monthly transfer to fresh medium. The abundance and diversity of total bacteria and fungi were monitored. Like LLDPE, lignin is a very complex polymer, so its biodegradation is closely linked to that of some recalcitrant plastics. For this reason, counting of ligninolytic microorganisms from the different enrichments was also performed. Additionally, the consortium members were isolated, molecularly identified and enzymatically characterized. The results revealed a loss of microbial diversity at each culture transfer at the end of the induced selection process. The consortium selected from selective enrichment in cultures with LLDPE in powder form was more effective compared to the consortium selected in cultures with LLDPE in film form, resulting in a reduction of microplastic weight between 2.5 and 5.5%. Some members of the consortia showed a wide range of enzymatic activities related to the degradation of recalcitrant plastic polymers, with Pseudomonas aeruginosa REBP5 or Pseudomonas alloputida REBP7 strains standing out. The strains identified as Castellaniella denitrificans REBF6 and Debaryomyces hansenii RELF8 were also considered relevant members of the consortia although they showed more discrete enzymatic profiles. Other consortium members could collaborate in the prior degradation of additives accompanying the LLDPE polymer, facilitating the subsequent access of other real degraders of the plastic structure. Although preliminary, the microbial consortia selected in this work contribute to the current knowledge of the degradation of recalcitrant plastics of anthropogenic origin accumulated in natural environments.

Characterization of Thermophilic Lignocellulolytic Microorganisms in Composting.

Composting involves the selection of a microbiota capable of resisting the high temperatures generated during the process and degrading the lignocellulose. A deep understanding of the thermophilic microbial community involved in such biotransformation is valuable to improve composting efficiency and to provide thermostable biomass-degrading enzymes for biorefinery. This study investigated the lignocellulose-degrading thermophilic microbial culturome at all the stages of plant waste composting, focusing on the dynamics, enzymes, and thermotolerance of each member of such a community. The results revealed that 58% of holocellulose (cellulose plus hemicellulose) and 7% of lignin were degraded at the end of composting. The whole fungal thermophilic population exhibited lignocellulose-degrading activity, whereas roughly 8-10% of thermophilic bacteria had this trait, although exclusively for hemicellulose degradation (xylan-degrading). Because of the prevalence of both groups, their enzymatic activity, and the wide spectrum of thermotolerance, they play a key role in the breakdown of hemicellulose during the entire process, whereas the degradation of cellulose and lignin is restricted to the activity of a few thermophilic fungi that persists at the end of the process. The xylanolytic bacterial isolates (159 strains) included mostly members of Firmicutes (96%) as well as a few representatives of Actinobacteria (2%) and Proteobacteria (2%). The most prevalent species were Bacillus licheniformis and Aeribacillus pallidus. Thermophilic fungi (27 strains) comprised only four species, namely Thermomyces lanuginosus, Talaromyces thermophilus, Aspergillus fumigatus, and Gibellulopsis nigrescens, of whom A. fumigatus and T. lanuginosus dominated. Several strains of the same species evolved distinctly at the stages of composting showing phenotypes with different thermotolerance and new enzyme expression, even not previously described for the species, as a response to the changing composting environment. Strains of Bacillus thermoamylovorans, Geobacillus thermodenitrificans, T. lanuginosus, and A. fumigatus exhibiting considerable enzyme activities were selected as potential candidates for the production of thermozymes. This study lays a foundation to further investigate the mechanisms of adaptation and acquisition of new traits among thermophilic lignocellulolytic microorganisms during composting as well as their potential utility in biotechnological processing.

Microbial communities of the olive mill wastewater sludge stored in evaporation ponds: The resource for sustainable bioremediation.

Olive Mill Wastewater (OMW) is a polluting residue from the olive oil industry. It is usually stored in open-air unprotected evaporation ponds where their sediments accumulate. This study compares the characteristics of OMW sludges stored for long-time in evaporation ponds and assesses their impact on the underlying soil layer. Physicochemical parameters, toxicity bioassays, and full characterization of the microbial community were analyzed. The extension of the polluting effects was assessed by analysis of toxicity, microbial biomass carbon, and respiration. Geostatistics was used to predict their spatial distribution. Organic matter and polyphenol content besides toxicity levels determine variations between OMW sludges and have a high impact on the microbiota they contain. The microbial community was abundant, diverse, and functionally active. However, the biodegradability of the sludges was hindered by the toxicity levels. Toxicity and biomass carbon were higher on the surface of the ponds than in the soil layer revealing a reduced leach flow and depletion of contaminants. The natural microbiota might be biostimulated by means of applying sustainable and feasible biological treatments in order to favor the OMW sludges bioremediation. These results open up the possibility of solving the environmental concern caused by its storage in similar scenarios, which are common in olive oil-producing countries.

Industrial Composting of Sewage Sludge: Study of the Bacteriome, Sanitation, and Antibiotic-Resistant Strains.

Wastewater treatment generates a huge amount of sewage sludge, which is a source of environmental pollution. Among the alternatives for the management of this waste, industrial composting stands out as one of the most relevant. The objective of this study was to analyze the bacterial population linked to this process and to determine its effectiveness for the reduction, and even elimination, of microorganisms and pathogens present in these organic wastes. For this purpose, the bacteriome and the fecal bacteria contamination of samples from different sewage sludge industrial composting facilities were evaluated. In addition, fecal bacteria indicators and pathogens, such as Salmonella, were isolated from samples collected at key stages of the process and characterized for antibiotic resistance to macrolide, ß-lactam, quinolone, and aminoglycoside families. 16S rRNA phylogeny data revealed that the process clearly evolved toward a prevalence of Firmicutes and Actinobacteria phyla, removing the fecal load. Moreover, antibiotic-resistant microorganisms present in the raw materials were reduced, since these were isolated only in the bio-oxidative phase. Therefore, industrial composting of sewage sludge results in a bio-safe final product suitable for use in a variety of applications.

Industrial composting of low carbon/nitrogen ratio mixtures of agri-food waste and impact on compost quality.

The agri-food waste (AW) require amendments for composting to adjust nutritional and physicochemical deficiencies. The theoretical mixtures formulation is difficult to reach on an industrial scale. The main objective of this work was to evaluate to what extent the composition of AW-based mixtures determines the quality of the final compost produced at the industrial scale. Raw materials having the same AW share characteristics, irrespectively of the amendments added, but their compost were different. All the materials were biological stable at the cooling phase, and mature enough at the end, although the degree of humification did not match with the absence of phytotoxicity. The final compost had sufficient quality even though the AW-based raw materials have a low C/N ratio (<20) and other characteristics such as high electrical conductivity (13 mS·cm-1) and pH (<8.5) that are unfavorable for composting. The management operations during industrial composting correct the deficiencies of raw materials.

Integral approach using bacterial microbiome to stabilize municipal solid waste.

Biological transformation of municipal solid waste is an environment-friendly management strategy against recalcitrant residues. The bacterial biome that inhabit said residues are responsible of decomposing both simple and complex materials. For this reason, processes such as composting, which favor the acceleration of the transformation of organic matter, can contribute to the degradation of municipal solid waste. Not only as mere fertilizer for crops, but also as methods for the recovery of solid waste. However, the control of the conditions necessary to achieve an optimal process on an industrial scale is a great concern. Thus, the aim of this work focuses on the characterization of the bacterial microbiome on three municipal solid waste facilities in order to deepen the role of microorganisms in the state of the final product obtained. For it, an intensive metagenomic analysis as well as a battery of physicochemical determinations were carried out. The lack of adequate thermophilic phases was decisive in finding certain bacterial genera, such as Lactobacillus, which was significant through these processes. Biodiversity did not follow a common pattern in the three processes, neither in abundance nor in richness but, in general, it was greater during the bio-oxidative stage. Despite the different trend in terms of the degradation of carbon fractions in these wastes, at the end of the biodegradation treatments, a sufficient degree of bioestabilization of the organic matter was reached. The results offer the opportunity to obtain a level of detail unprecedented of the structure, dynamics and function of the bacterial community in real conditions, without the control offered by laboratory conditions or pilot plants.

Bioremediation of Olive Mill Wastewater sediments in evaporation ponds through in situ composting assisted by bioaugmentation.

The common method for the disposal of olive oil mill wastewater (OMW) has been its accumulation in evaporation ponds where OMW sediments concentrate. Due to the phytotoxic and antimicrobial effect of OMW, leaks from ponds can pollute soils and water bodies. This work focuses on the search for microorganisms that can be used as inocula for bioremediation of polluted matrices in OMW ponds by means of in situ composting. Two fungi isolated from OMW sediments, Aspergillus ochraceus H2 and Scedosporium apiospermum H16, presented suitable capabilities for this use as a consortium. Composting eliminated the phyto- and ecotoxicity of OMW sediments by depleting their main toxic components. Inoculation with the fungal consortium improved the bioremediation efficacy of the technique by hastening the decrease of phytotoxicity and ecotoxicity and enhancing phytostimulant property of compost produced. This procedure constitutes a promising strategy for bioremediation of OMW polluted sites.

Biodiversity and succession of mycobiota associated to agricultural lignocellulosic waste-based composting.

A comprehensive characterization of the culturable mycobiota associated to all stages of lignocellulose-based composting was achieved. A total of 77 different isolates were detected, 69 of which were identified on the basis of the 5.8-ITS region sequencing. All the isolates were assigned to the phyla Ascomycota and Basidiomycota, with prevalence of the Sordariomycetes (19) and Eurotiomycetes (17) classes. Penicillium was the most represented genus (11 species), while the species Gibellulopsis nigrescens and Microascus brevicaulis were detected at all the composting stages and showed the highest relative abundances. Fungal diversity decreased as the process proceed, while similarity between fungal communities associated to different samples were maximal for those phases closely connected chronologically and showing similar biological activity degree. Thus, the structure of the lignocellulose-based composting mycobiota can be divided into two major stages corresponding to bio-oxidative phase and maturation phase together with the final product, with a transitional cooling stage joining both of them.

Enzymatic characterization of microbial isolates from lignocellulose waste composting: chronological evolution.

Successful composting is dependent upon microbial performance. An interdependent relationship is established between environmental and nutritional properties that rule the process and characteristics of the dominant microbial communities. To reach a better understanding of this relationship, the dynamics of major metabolic activities associated with cultivable isolates according to composting phases were evaluated. Ammonification (72.04%), amylolysis (35.65%), hemicellulolyis (30.75%), and proteolysis (33.61%) were the more frequent activities among isolates, with mesophilic bacteria and fungi as the prevalent microbial communities. Bacteria were mainly responsible for starch hydrolysis, while a higher percentage of hemicellulolytic and proteolytic isolates were ascribable to fungi. Composting seems to exert a functional selective effect on microbial communities by promoting the presence of specific metabolically dominant groups at each stage of the process. Moreover, the application of conglomerate analysis led to the statement of a clear correlation between the chronology of the process and characteristics of the associated microbiota. According to metabolic capabilities of the isolates and their density, three clear clusters were obtained corresponding to the start of the process, including the first thermophilic peak, the rest of the bio-oxidative stage, and the maturation phase.

Exploiting composting biodiversity: study of the persistent and biotechnologically relevant microorganisms from lignocellulose-based composting.

The composting ecosystem is a suitable source for the discovery of novel microorganisms and secondary metabolites. This work analyzes the identity of microbial community that persists throughout lignocellulose-based composting, evaluates their metabolic activities and studies the capability of selected isolates for composting bioaugmentation. Bacterial species of the phyla Firmicutes, Actinobacteria and Proteobacteria and fungi of the phylum Ascomycota were ubiquitous throughout the composting. The species Arthrobacter russicus, Microbacterium gubbeenense, Ochrocladosporium frigidarii and Cladosporium lignicola are detected for the first time in this ecosystem. In addition, several bacterial and fungal isolates exhibited a wide range of metabolic capabilities such as polymers (lignocellulose, protein, lipids, pectin and starch) breakdown and phosphate-solubilization that may find many biotechnological applications. In particular, Streptomyces albus BM292, Gibellulopsis nigrescens FM1397 and FM1411, Bacillus licheniformis BT575, Bacillus smithii AT907 and Alternaria tenuissima FM1385 exhibited a great potential as inoculants for composting bioaugmentation.

Tracking organic matter and microbiota dynamics during the stages of lignocellulosic waste composting.

The dynamics of biologically meaningful soluble and polymeric carbon fractions and the combined relationships between physical, chemical and biological parameters during composting of lignocellulosic waste were evaluated. The first thermophilic stage is crucial in determining the further evolution of soluble and polymeric carbon fractions but the dynamics of carbon is still important at the maturation stage. Multivariate data analysis showed that not only are all parameters interrelated but also influence one another's variability. To discern completion of bio-oxidative stage other parameters in addition to temperature should be measured. Evaluation of soluble organic carbon, microbial biomass carbon, pH and inorganic nitrogen can be of great use in detecting the composting stage. This study offers new insights into the mechanisms involved in the biodegradation of organic matter and help to prioritize the parameters that contribute at critical stages of the process.

Ecotoxicity and fungal deterioration of recycled polypropylene/wood composites: effect of wood content and coupling.

Recycled polypropylene (rPP) was recovered from an industrial shredder and composites were prepared with a relatively wide range of wood content and with two coupling agents, a maleated PP (MAPP) and a maleated ethylene-propylene-diene elastomer (MAEPDM). The mechanical properties of the composites showed that the coupling agents change structure only slightly, but interfacial adhesion quite drastically. The durability of the materials was determined by exposing them to a range of fungi and, ecotoxicity was studied on the aquatic organism Vibrio fischeri. The composites generally exhibit low acute toxicity, with values below the levels considered to have direct ecotoxic effect on aquatic ecosystems (<2 toxic units). Their toxicity to V. fischeri depended on the presence of the coupling agents with larger E50 values in 24-h aqueous extracts from composites containing MAPP or MAEPDM in comparison to composites without any coupling agent. Evaluation of resistance against fungal colonization and deterioration proved that wood facilitates fungal colonization. Fungi caused slight mass loss (below 3%) but it was not correlated with substantial deterioration in material properties. MAPP seems to be beneficial in the retention of mechanical properties during fungal attack. rPP/wood composites can be considered non-ecotoxic and quite durable, but the influence of wood content on resistance to fungal attack must be taken into account for materials intended for applications requiring long-term outdoor exposure.

Compost as a source of microbial isolates for the bioremediation of heavy metals: in vitro selection.

Heavy metal pollution has become a major environmental concern nowadays and the bioremediation of polluted habitats is an increasingly popular strategy due to both its efficiency and safety. A screening and selection protocol based on different composting processes was designed in order to isolate heavy metal-resistant microorganisms. A collection of 51 microorganisms was obtained and most of them showed the capability to tolerate heavy metals in multi-polluted aqueous systems (Cd(II), Cr(VI), Ni, Pb, Zn(II)), as well as to remove them. The highest detoxification ratios were observed for Pb. Some of the isolates detoxifying more than a 90% of this metal, while the other metals were removed in a range between 20% and 60%. The best isolates (Graphium putredinis, Fusarium solani, Fusarium sp. and Penicillium chrysogenum) were further assayed in order to determine the predominant removal mechanism and the potential use of their dead biomass as a biosorbent. Intracellular accumulation was the prevalent mechanism for most isolates and metals, with the exception of Ni. In this case, the proportion removed by extracellular adsorption was similar or even higher than that removed by intracellular accumulation. Thus, the efficiency of living cells was higher than that of dead biomass except in the case of Ni.