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Although several works have been performed from an engineering point of view, a limited number of studies have focused on microbial communities involved in the humification of aged landfill leachates. In this work, cultivation techniques, next-generation sequencing, and phospholipid fatty acid analysis were adopted to decrypt the diversity and the ecophysiological properties of the dominant microbiota in aged landfill leachate. Based on Illumina sequencing, Betaproteobacteria, Bacteroidetes, Actinobacteria, and Alphaproteobacteria dominated the aged landfill leachate. The main taxa identified at genus level were Pusillimonas-like bacteria and Leucobacter (41.46% of total reads), with all of them being also isolated through cultivation. The presence of Pusillimonas-like bacteria was also verified by the detection of cyclo17:0 and iso-19:0 fatty acids in aged landfill leachate microbiota. Despite that almost all bacterial isolates exhibited extracellular lipolytic ability, no particular specificity was observed in the type of substrate utilized. The prevalence of effective degraders, such as Pusillimonas-like bacteria, makes the aged landfill leachate an ideal source for isolation of novel microorganisms with potential in situ bioremediation uses.

Mature landfill leachate is a heavily-polluted wastewater due to its recalcitrant nature of organic matter, and high ammonia and salt content. Despite the moderate saline and alkaline nature of this habitat, no attention has been paid to the isolation and functional role of extremophiles in such environment. In this work, a total of 73 and 29 bacterial strains were isolated by using alkaline and saline media, respectively, while bacteria from mature landfill leachate growing in these media were enumerated as 1.5 ± 0.1 (×10) and 5.8 ± 0.9 (×10) cfu/L. Based on their pH and salt ranges and optima for growth, all bacterial isolates were halotolerant alkaliphiles (either facultative or obligate), with the majority of them being extremely halotolerant bacteria. These halotolerant alkaliphiles were classified into 14 operational taxonomic units (OTUs). Of these, 12 are placed within known halophilic and alkaliphilic species of the genera Dietzia, Glycocaulis, Halomonas, Marinobacter, Piscibacillus and Rhodobacter, while the remaining OTUs represented two novel phylogenetic linkages among the families Cyclobacteriaceae and Rhodobacteraceae. Examination of their hydrolytic ability through the performance of lipase, protease and β-glucosidase assays using landfill leachate as the growth substrate revealed that all halotolerant alkaliphiles isolated exhibited extremely high lipolytic activities (up to 78,800 U g protein), indicating a key involvement of extremophilic microbiota at the late landfill maturation stage. The wide extremely lipolytic halotolerant alkaliphilic community identified also makes mature landfill leachate an ideal microbial pool for the isolation of novel extremophiles of biotechnological interest.

A membrane bioreactor was operated under intermittent aeration and various organic loading rates (OLR: 0.070, 0.159 and 0.291 g COD L d) to remove carbon and nitrogen from mature landfill leachate, where external carbon source (glycerol) addition resulted in effective nitrate removal. A relative increase in soluble microbial product (SMP) over extracellular polymeric substances (EPS) was observed at the highest OLR and glycerol addition, whereas no membrane biofouling occurred. SMP (proteins and carbohydrates) and carbohydrate EPS correlated positively and negatively, respectively, with suspended solids and transmembrane pressure (TMP). Moreover, proteinous SMP significantly correlated with carbon and nitrogen load. Principal component analysis also revealed the influence of leachate organic and nitrogen content on biomass production, TMP and sessile ciliate densities. Although filamentous index (FI) was sustained at high levels (3-4), with Haliscomenobacter hydrossis being the main filamentous bacterium identified, no bulking phenomena occurred. High glycerol addition resulted in a rapid increase in sessile ciliate population. Increased Epistylis and Vorticella microstoma population was detected by microscopic examination during high glycerol addition, while a remarkable Rhogostoma population (supergroup Rhizaria) was identified by molecular techniques. The contribution of Rhizaria in nitrogen processes may lead to the dominance of Rhogostoma during landfill leachate treatment.

Despite that diclofenac has been embodied to the European watch list of priority substances of concern, studies on diclofenac biodegradation are limited and the diversity of diclofenac-degrading microbiota remains unknown. In this work, an immobilized cell biofilter was constructed and operated to evaluate its effectiveness to depurate high strength diclofenac wastewater and to identify the diclofenac-degrading community accommodated in activated sludge by employing high-throughput sequencing techniques. After a two-month adaptation period, biofilter removal efficiencies reached values as high as 97.63 ± 0.62%, whereas utilization of diclofenac in the immobilized cell biofilter led to a drastic pH decrease. Based on Illumina sequencing, the major bacterial taxa identified in the immobilized cell biofilter were members of the species Granulicella pectinivorans and Rhodanobacter terrae, followed by members of the species Castellaniella denitrificans, Parvibaculum lavamentivorans, Bordetella petrii, Bryocella elongata and Rhodopseudomonas palustris. The ability of such taxa to utilize a wide range of carbon sources and to effectively adapt under acidic conditions seemed to be the main parameters, which favored their prevalence in the immobilized cell biofilter. In addition, Wickerhamiella was the predominant fungal taxon in the immobilized cell biofilter, which appears to be actively involved in diclofenac degradation in activated sludge systems.

The orange juice processing sector produces worldwide massive amounts of waste, which is characterized by high lignin, cellulose and hemicellulose content, and which exceeds 40% of the fruit’s dry weight (d.w.). In this work, the diversity and the biotechnological potential of xylan-degrading microbiota in orange juice processing waste were investigated through the implementation of an enrichment isolation strategy followed by enzyme assays for the determination of xylanolytic activities, and via next generation sequencing for microbial diversity identification. Intracellular rather than extracellular endo-1,4-β-xylanase activities were detected, indicating that peripheral cell-bound (surface) xylanases are involved in xylan hydrolysis by the examined microbial strains. Among the isolated microbial strains, bacterial isolates belonging to Pseudomonas psychrotolerans/P. oryzihabitans spectrum (99.9%/99.8% similarity, respectively) exhibited activities of 280 U/mg protein. In contrast, almost all microbial strains isolated exerted low extracellular 1,4-β-xylosidase activities (<5 U/mg protein), whereas no intracellular 1,4-β-xylosidase activities were detected for any of them. Illumina data showed the dominance of lactic and acetic acid bacteria and of the yeasts Hanseniaspora and Zygosaccharomyces. This is the first report on indigenous xylanolytic microbiota isolated from orange juice processing waste, possessing the biotechnological potential to serve as biocatalysts for citrus biomass valorization through the production of high-added value products and energy recovery.

Despite the fact that biological nitrogen removal (BNR) process has been studied in detail in laboratory- and pilot-scale sequencing batch reactor (SBR) systems treating landfill leachate, a limited number of research works have been performed in full-scale SBR plants regarding nitrification and denitrification. In the current study, a full-scale twin SBR system in series of 700 m (350 m each) treating medium-age landfill leachate was evaluated in terms of its carbon and nitrogen removal efficiency in the absence and presence of external carbon source, i.e., glycerol from biodiesel production. Both biodegradable organic carbon and ammonia were highly oxidized [biochemical oxygen demand (BOD) and total Kjehldahl nitrogen (TKN) removal efficiencies above 90%], whereas chemical oxygen demand (COD) removal efficiency was slightly above 40%, which is within the range reported in the literature for pilot-scale SBRs. As the consequence of the high recalcitrant organic fraction of the landfill leachate, dissimilatory nitrate reduction was restricted in the absence of crude glycerol, although denitrification was improved by electron donor addition, resulting in TN removal efficiencies above 70%. Experimental data revealed that the second SBR negligibly contributed to BNR process, since carbon and ammonia oxidation completion was achieved in the first SBR. On the other hand, the low VSS/SS ratio, due to the lack of primary sedimentation, highly improved sludge settleability, resulting in sludge volume indices (SVI) below 30 mL g.