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.

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.