Microbial Community Structure among Honey Samples of Different Pollen Origin.

Honey's antibacterial activity has been recently linked to the inhibitory effects of honey microbiota against a range of foodborne and human pathogens. In the current study, the microbial community structure of honey samples exerting pronounced antimicrobial activity was examined. The honey samples were obtained from different geographical locations in Greece and had diverse pollen origin (fir, cotton, fir-oak, and Arbutus unedo honeys). Identification of honey microbiota was performed by high-throughput amplicon sequencing analysis, detecting 335 distinct taxa in the analyzed samples. Regarding ecological indices, the fir and cotton honeys possessed greater diversity than the fir-oak and Arbutus unedo ones. Lactobacillus kunkeei (basionym of Apilactobacillus kun-keei) was the predominant taxon in the fir honey examined. Lactobacillus spp. appeared to be favored in honey from fir-originated pollen and nectar since lactobacilli were more pronounced in fir compared to fir-oak honey. Pseudomonas, Streptococcus, Lysobacter and Meiothermus were the predominant taxa in cotton honey, whereas Lonsdalea, the causing agent of acute oak decline, and Zymobacter, an osmotolerant facultative anaerobic fermenter, were the dominant taxa in fir-oak honey. Moreover, methylotrophic bacteria represented 1.3-3% of the total relative abundance, independently of the geographical and pollen origin, indicating that methylotrophy plays an important role in honeybee ecology and functionality. A total of 14 taxa were identified in all examined honey samples, including bacilli/anoxybacilli, paracocci, lysobacters, pseudomonads, and sphingomonads. It is concluded that microbial constituents of the honey samples examined were native gut microbiota of melliferous bees and microbiota of their flowering plants, including both beneficial bacteria, such as potential probiotic strains, and animal and plant pathogens, e.g., Staphylococcus spp. and Lonsdalea spp. Further experimentation will elucidate aspects of potential application of microbial bioindicators in identifying the authenticity of honey and honeybee-derived products.

Retrofitting a full-scale multistage landfill leachate treatment plant by introducing coagulation/flocculation/sedimentation and ultrafiltration process steps.

Considering that landfilling still remains among the most commonly used methods for the confrontation of solid wastes, effective methods should be applied to treat the leachate generated, due to its recalcitrant nature. In this work, a full-scale system consisting of two SBRs operating in parallel (350 m3 each) and two activated carbon (AC) columns operating in series (3 m3 each) was retrofitted by introducing a coagulation/flocculation/sedimentation (C/F/S) unit of 7.8 m3 and an ultrafiltration (UF) membrane of 100 m2 to effectively treat landfill leachate. The raw leachate was characterized by high COD and NH4+-N concentration, i.e., 3095 ± 706 mg/L and 1054 ± 141 mg/L respectively, a BOD/COD ratio of 0.22, and high concentrations of certain heavy metals. Leachate processing in this retrofitted multistage treatment system resulted in total COD removal efficiency of 89.84%, with biological treatment, C/F, UF, and AC contributing 46.31%, 4.68%, 15.98%, and 22.87% to the overall organic content removal. The retrofitted scheme achieved an overall NH4+-N and TKN removal of 92.03% and 91.75% respectively, attributed mostly to the activity of an effective nitrifying community. Color number (CN) was reduced by 26.96%, 10.29%, 15.94%, and 5.39% after the activated sludge, the C/F, the UF, and the AC adsorption process respectively, corresponding to a 58.91% overall decrease. Regarding heavy metal removal, all elements examined, apart from Ni, i.e., effluent As, Cd, Co, Cr, Cu, Hg, Mg, Mn, and Pb, were below the legislative limits set by the national authorities for restricted or unrestricted irrigation. Lastly, total operating expenses (OPEX) were estimated as equal to 72,687 €/year or 6.64 €/m3.

Biotreatment efficiency, degradation mechanism and bacterial community structure in an immobilized cell bioreactor treating triclosan-rich wastewater.

Biotreatment of triclosan is mainly performed in conventional activated sludge systems, which, however, are not capable of completely removing this antibacterial agent. As a consequence, triclosan ends up in surface and groundwater, constituting an environmental threat, due to its toxicity to aquatic life. However, little is known regarding the diversity and mechanism of action of microbiota capable of degrading triclosan. In this work, an immobilized cell bioreactor was setup to treat triclosan-rich wastewater. Bioreactor operation resulted in high triclosan removal efficiency, even greater than 99.5%. Nitrogen assimilation was mainly occurred in immobilized biomass, although nitrification was inhibited. Based on Illumina sequencing, Bradyrhizobiaceae, followed by Ferruginibacter, Thermomonas, Lysobacter and Gordonia, were the dominant genera in the bioreactor, representing 38.40 ± 0.62% of the total reads. However, a broad number of taxa (15 genera), mainly members of Xanthomonadaceae, Bradyrhizobiaceae and Chitinophagaceae, showed relative abundances between 1% and 3%. Liquid Chromatography coupled to Quadrupole Time-Of-Flight Mass Spectrometry (LC-QTOF-MS) resulted in the identification of catabolic routes of triclosan in the immobilized cell bioreactor. Seven intermediates of triclosan were detected, with 2,4-dichlorophenol, 4-chlorocatechol and 2-chlorohydroquinone being the key breakdown products of triclosan. Thus, the immobilized cell bioreactor accommodated a diverse bacterial community capable of degrading triclosan.

Activated sludge microbial communities and hydrolytic potential in a full-scale SBR system treating landfill leachate.

Landfill leachate, due to its recalcitrant nature and toxicity, poses a serious environmental threat, which requires the implementation of effective treatment processes. In this work, a full-scale treatment system consisting of two Sequencing Batch Reactors (SBRs) was used for the processing of landfill leachate of intermediate to mature age (BOD/COD ratio of 0.16). Biosystem operation resulted in BOD5, COD and TKN removal efficiencies of 81%, 39% and 76%, respectively, whereas the low residual NO3--N concentration in the effluent (4.01 ± 0.10 mg/L) was indicative of the efficient denitrification process. Assessment of hydrolytic potential of activated sludge revealed high endocellular and extracellular lipase activities, which reached values up to 206 and 141 U/g protein respectively, possibly as the consequence of plastics degradation during maturation process. Implementation of Illumina sequencing indicated the predominance of Alphaproteobacteria, accompanied by members of Bacteroidetes, Betaproteobacteria and Chloroflexi. Paracoccus was the predominant genus identified, followed by representatives of the genera Bellilinea, Flavobacterium, Thauera and Truepera. Nitrosomonas was the major ammonia-oxidizing bacterium (AOB), while nitrite oxidation was mainly achieved by the uncultured nitrite-oxidizing bacterium (NOB) Candidatus Nitrotoga.

Dynamics of the Gut Bacteriome During a Laboratory Adaptation Process of the Mediterranean Fruit Fly, Ceratitis capitata.

Laboratory adaptation process used in sterile insect technique (SIT) programs can exert a significant impact on the insect-gut microbiome relationship, which may negatively impact the quality and performance of the fly. In the present study, changes in the gut microbiota that occur through laboratory adaptation of two Ceratitis capitata populations were investigated: Vienna 8 genetic sexing strain (GSS), a long-established control line, and a wild population recently introduced to laboratory conditions. The bacterial profiles were studied for both strains using amplicon sequencing of the 16S rRNA V3-V4 hypervariable region in larvae and in the gastrointestinal tract of teneral (1 day) and adults (5 and 15 days) reared under laboratory conditions for 14 generations (F0-F13). Findings demonstrated the development of distinct bacterial communities across the generations with differences in the bacterial composition, suggesting a strong impact of laboratory adaptation on the fly bacteriome. Moreover, different bacterial profiles were observed between wild and Vienna 8 FD-GSS displaying different patterns between the developmental stages. Proteobacteria, mainly members of the Enterobacteriaceae family, represented the major component of the bacterial community followed by Firmicutes (mainly in Vienna 8 FD-GSS adults) and Chlamydiae. The distribution of these communities is dynamic across the generations and seems to be strain- and age-specific. In the Vienna 8 FD-GSS population, Providencia exhibited high relative abundance in the first three generations and decreased significantly later, while Klebsiella was relatively stable. In the wild population, Klebsiella was dominant across most of the generations, indicating that the wild population was more resistant to artificial rearing conditions compared with the Vienna 8 FD-GSS colony. Analysis of the core bacteriome revealed the presence of nine shared taxa between most of the examined medfly samples including Klebsiella, Providencia, Pantoea, and Pseudomonas. In addition, the operational taxonomic unit co-occurrence and mutual exclusion networks of the wild population indicated that most of the interactions were classified as co-presence, while in the Vienna 8 FD-GSS population, the number of mutual exclusions and co-presence interactions was equally distributed. Obtained results provided a thorough study of the dynamics of gut-associated bacteria during the laboratory adaptation of different Ceratitis capitata populations, serving as guidance for the design of colonization protocols, improving the effectiveness of artificial rearing and the SIT application.

Evaluation of electrochemical and O3/UV/H2O2 methods at various combinations during treatment of mature landfill leachate.

In this study, electrochemical treatment and application of O3/UV/H2O2 in various combinations were evaluated in respect to their efficiency to depurate mature landfill leachate. Based on preliminary experiments, electrochemical treatment using stainless-steel electrodes at 2 cm gap was performed optimally at 50 mA/cm2 and pH 6, while application of O3 at 120 L/h, UV at 991 J/cm2 and H2O2 concentration of 1 g/L was carried out. Electrochemical treatment and O3/UV/H2O2 under optimal conditions were applied as follows: I) electrochemical treatment, followed by O3/UV/H2O2 and solids precipitation, II) electrochemical treatment, followed by precipitation and then by O3/UV/H2O2 treatment, and III) O3/UV/H2O2, followed by electrochemical treatment. A low performance was observed when O3/UV/H2O2 preceding electrochemical treatment. Solids, TKN and total COD (tCON) removal was primarily achieved through electrocoagulation, whereas color and soluble COD (sCOD) reduction was mainly attributed to electrochemical oxidation. Experimental setup I was the most efficient treatment scheme, resulting in tCOD, sCOD, TKN, TSS, SACUV254nm and color number reduction of 73%, 80%, 76%, 79%, 94% and 98%, respectively. Indeed, O3/UV/H2O2 step could be omitted since its effectiveness was restricted during landfill leachate treatment. In conclusion, electrochemical treatment followed by precipitation could result in effective reduction of nutrients and color.

Effect of Chlorination on Microbiological Quality of Effluent of a Full-Scale Wastewater Treatment Plant.

The evaluation of effluent wastewater quality mainly relies on the assessment of conventional bacterial indicators, such as fecal coliforms and enterococci; however, little is known about opportunistic pathogens, which can resist chlorination and may be transmitted in aquatic environments. In contrast to conventional microbiological methods, high-throughput molecular techniques can provide an accurate evaluation of effluent quality, although a limited number of studies have been performed in this direction. In this work, high-throughput amplicon sequencing was employed to assess the effectiveness of chlorination as a disinfection method for secondary effluents. Common inhabitants of the intestinal tract, such as Bacteroides, Arcobacter and Clostridium, and activated sludge denitrifiers capable of forming biofilms, such as Acidovorax, Pseudomonas and Thauera, were identified in the chlorinated effluent. Chloroflexi with dechlorination capability and the bacteria involved in enhanced biological phosphorus removal, i.e., Candidatus Accumulibacter and Candidatus Competibacter, were also found to resist chlorination. No detection of Escherichia indicates the lack of fecal coliform contamination. Mycobacterium spp. were absent in the chlorinated effluent, whereas toxin-producing cyanobacteria of the genera Anabaena and Microcystis were identified in low abundances. Chlorination significantly affected the filamentous bacteria Nocardioides and Gordonia, whereas Zoogloea proliferated in the disinfected effluent. Moreover, perchlorate/chlorate- and organochlorine-reducing bacteria resisted chlorination.

Biotreatment efficiency, hydrolytic potential and bacterial community dynamics in an immobilized cell bioreactor treating caper processing wastewater under highly saline conditions.

Although caper processing wastewaters (CPW) are characterized by high organic content and salt concentration, no attempt has been made to treat these effluents. In this study, an immobilized cell bioreactor efficiently treated CPW even at hypersaline conditions (100 g/L salinity). Nitrogen was mainly assimilated during biotreatment, as nitrification was inhibited at elevated salinities. The hydrolytic potential was assessed by determining glucanase, xylanase, glucosidase, lipase and protease activities, which were negatively affected above 20 g/L salinity as the consequence of the inhibition of non-halotolerant microbiota. Succession of non-halotolerant taxa by the slightly halotolerant bacteria Defluviimonas, Amaricoccus, Arenibacter, Formosa and Muricauda, and then by the moderately/extremely halotolerant genera Halomonas, Roseovarius and Idiomarina occurred over salinity increase. Diversity indices were reduced during transition from moderately saline to hypersaline conditions. A distinct network was formed at hypersaline conditions, consisting of the halotolerant genera Halomonas, Idiomarina, Saliterribacillus and Gracilibacillus.

Optimization aspects of the biological nitrogen removal process in a full-scale twin sequencing batch reactor (SBR) system in series treating landfill leachate.

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 m3 (350 m3 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 (BOD5) 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-1.

Dominance of candidate Saccharibacteria in a membrane bioreactor treating medium age landfill leachate: Effects of organic load on microbial communities, hydrolytic potential and extracellular polymeric substances.

A membrane bioreactor (MBR), accomplishing high nitrogen removal efficiencies, was evaluated under various landfill leachate concentrations (50, 75 and 100% v/v). Proteinous and carbohydrate extracellular polymeric substances (EPS) and soluble microbial product (SMP) were strongly correlated (p<0.01) with organic load, salinity and NH4+-N. Exceptionally high ß-glucosidase activities (6700-10,100Ug-1) were determined during MBR operation with 50% v/v leachate, as a result of the low organic carbon availability that extendedly induced ß-glucosidases to breakdown the least biodegradable organic fraction. Illumina sequencing revealed that candidate Saccharibacteria were dominant, independently of the leachate concentration applied, whereas other microbiota (21.2% of total reads) disappeared when undiluted leachate was used. Fungal taxa shifted from a Saccharomyces- to a newly-described Cryptomycota-based community with increasing leachate concentration. Indeed, this is the first report on the dominance of candidate Saccharibacteria and on the examination of their metabolic behavior in a bioreactor treating real wastewater.

Bacterial community structure and prevalence of Pusillimonas-like bacteria in aged landfill leachate.

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.

Protozoan indicators and extracellular polymeric substances alterations in an intermittently aerated membrane bioreactor treating mature landfill leachate.

A membrane bioreactor was operated under intermittent aeration and various organic loading rates (OLR: 0.070, 0.159 and 0.291 g COD L-1 d-1) 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.

Novel hydrolytic extremely halotolerant alkaliphiles from mature landfill leachate with key involvement in maturation process.

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 (×108) and 5.8 ± 0.9 (×108) 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 ß-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-1 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.

Effects of high organic load on amoA and nirS gene diversity of an intermittently aerated and fed membrane bioreactor treating landfill leachate.

The effects of external carbon source addition on the nitrification and denitrification process were investigated in an intermittently aerated and fed membrane bioreactor treating landfill leachate by recording system performance, and amoA and nirS diversity dynamics using pyrosequencing. By adding 950mg/L glycerol, denitrification was optimized, resulting in total nitrogen removal efficiency of 81.0±2.4%. Under these conditions, amoA diversity was dominated by genotypes related to Nitrosomonas europaea, while increase in leachate's content and in glycerol addition by 50% led to irreversible inhibition of nitrification and enhanced ammonia accumulation, causing a severe suppression of Nitrosomonas and an increase in the relative abundance of Nitrosospira. However, this increase not only affected ammonia oxidizers, but also caused a massive shift in denitrifying community structure, resulting in the suppression of Arenimonas metalli-, Candidatus Accumulibacter- and Sulfuritalea hydrogenivorans-nirS related genotypes and the predominance of nirS-associated with Acidovorax and Thaurea sp.