First Record of Neofusicoccum luteum and Colletotrichum gloeosporioides Causing Anthracnose and Stem End Rot on Avocado Fruits in Greece.

In May 2022, rot symptoms were observed 5 days after storage on fresh avocado fruits cv "Lamb Hass" harvested from a 3.4 ha organic orchard in Chania, Crete exhibiting 30% symptom incidence. Brownish-green sunken lesions and soft rot with dark brown lesions covering up to 50% of the mesocarp on fruits and blackish soft lesions on fruit stem ends were observed. To isolate the pathogens, fruits were surface sterilized using 1% NaOCl for 1 min, placed in 70% ethanol for 30 s and washed twice with sterile distilled water. Then, small pieces were excised from the fruit rot margins and transferred on PDA amended with 0.015% streptomycin-sulfate. Single-spore isolates were incubated on PDA for 10 days and subjected to morphological examination. Two distinct pathogenic fungal isolates were obtained, and their symptoms were re-examined on avocado fruits. The first isolate (A1) obtained from the fruit stem end, initially produced hyaline dense aerial mycelia, being gray and black on the upper and lower surface of the Petri dishes, respectively. The second isolate (A2) obtained from the main body of the fruit, formed round, grayish colonies, with orange conidial aggregates. Based on morphological characteristics (Phillips et al.,2013; Weir et al., 2012), isolates were preliminary identified as Neofusicoccum sp. (A1) and Colletotrichum sp. (A2). Isolates were molecularly identified by sequencing of the ITS-5.8S rRNA, translation elongation factor 1-alpha (tef1) and beta-tubulin (tub2) genes. PCRs were conducted using primer pairs ITS4/ITS5, EF1-728F/EF1986R and Bt2a/Bt2b as well as ITS4/ITS5 and 5'-tef1/3'-tef1 and Bt2a/Bt2b for isolates A1 and A2, respectively (Carbone & Kohn, 1999; Glass & Donaldson, 1995; Rojas et al., 2010; Weir et al., 2012; White et al., 1990). The sequences were deposited into GenBank under the accession numbers OQ852465, OQ867962, OQ867965 for N. luteum and, OQ852466, OQ867963 and OQ867964 for C. gloeosporioides. Based on Multilocus sequence analysis (MLSA), a phylogenetic tree was constructed using concatenated sequences, following Kimura's two parameter model (1980), which confirmed their identity as N luteum and C. gloeosporioides strains. Mature avocado fruits (cv. Hass) were surface sterilized and dried. Consequently, incised fruits were inoculated with mycelial agar plugs (5 mm in diameter) cut from the edge of rapidly growing colonies of N. luteum and C. gloeosporioides strains. Fruits incubated in moist chambers and at 25°C for 5 days in the dark. Fruit bodies and stems were inoculated with the respective isolates and sterile agar plugs in the case of the control. Five fruits were used for each pathogenic trial per fungal isolate, which was repeated twice. After symptom occurrence, these pathogenic isolates were re-isolated successfully and molecularly identified, while exhibiting similar to original symptoms confirming Koch's postulates. While other reports exist on the presence of these pathogens in different countries worldwide, this is the first report of C. gloeosporioides and N. luteum as post-harvest pathogens of avocado, which is an economically important crop of Crete, in Greece (Akgül et al., 2016). This study provides the means for the accurate identification of these fungal pathogens causing avocado fruit rots and taking into consideration the available treatment options can contribute to establishing effective management strategies.

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.

Biochemical and nutritional characterization of the medfly gut symbiont Enterobacter sp. AA26 for its use as probiotics in sterile insect technique applications.

BACKGROUND: Enterobacter sp. AA26 was recently isolated from the midgut of Ceratitis capitata (Wiedemann) and it was shown to have positive effects in rearing efficiency when used as larval probiotics. In this study, biomass production was carried out in bench-scale bioreactors to elucidate the biokinetic properties of Enterobacter sp. AA26 and its nutritional value. RESULTS: Strain AA26 is a psychrotolerant, halotolerant, facultatively anaerobic bacterium with broad pH range for growth (pH 4 to 10.2), which possessed the typical biochemical profile of Enterobacter spp. The specific oxygen uptake rate (SOUR) was calculated as 63.2 ± 1.26 and 121 ± 1.73 mg O2 g- 1 VSS h- 1, with the yield coefficients in acetate and glucose being equal to 0.62 ± 0.03 and 0.67 ± 0.003 g biomass produced/g substrate consumed, respectively. The maximum specific growth rate (µmax) of strain AA26 grown in fill-and-draw bioreactors at 20 °C and 35 °C was 0.035 and 0.069 h- 1, respectively. Strain AA26 grew effectively in agro-industrial wastewaters, i.e. cheese whey wastewater (CWW), as alternative substrate for replacing yeast-based media. Biomass of strain AA26 could provide all the essential amino acids and vitamins for the artificial rearing of C. capitata. Greater intracellular α- and ß-glucosidase activities were observed during growth of strain AA26 in CWW than in yeast-based substrate, although the opposite pattern was observed for the respective extracellular activities (p < 0.01). Low protease activity was exhibited in cells grown in yeast-based medium, while no lipase activities were detected. CONCLUSIONS: The ability of strain AA26 to grow in agro-industrial wastes and to provide all the essential nutrients can minimize the cost of commercial media used for mass rearing and large scale sterile insect technique applications.

Enterobacter sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications.

BACKGROUND: Insect species have established sophisticated symbiotic associations with diverse groups of microorganisms including bacteria which have been shown to affect several aspects of their biology, physiology, ecology and evolution. In addition, recent studies have shown that insect symbionts, including those localized in the gastrointestinal tract, can be exploited for the enhancement of sterile insect technique (SIT) applications against major insect pests such as the Mediterranean fruit fly (medfly) Ceratitis capitata. We previously showed that Enterobacter sp. AA26 can be used as probiotic supplement in medfly larval diet improving the productivity and accelerating the development of the VIENNA 8 genetic sexing strain (GSS), which is currently used in large scale operational SIT programs worldwide. RESULTS: Enterobacter sp. AA26 was an adequate nutritional source for C. capitata larvae, comprising an effective substitute for brewer's yeast. Incorporating inactive bacterial cells in the larval diet conferred a number of substantial beneficial effects on medfly biology. The consumption of bacteria-based diet (either as full or partial yeast replacement) resulted in decreased immature stages mortality, accelerated immature development, increased pupal weight, and elongated the survival under stress conditions. Moreover, neither the partial nor the complete replacement of yeast with Enterobacter sp. AA26 had significant impact on adult sex ratio, females' fecundity, adults' flight ability and males' mating competitiveness. The absence of both yeast and Enterobacter sp. AA26 (deprivation of protein source and possible other important nutrients) from the larval diet detrimentally affected the larval development, survival and elongated the immature developmental duration. CONCLUSIONS: Enterobacter sp. AA26 dry biomass can fully replace the brewer's yeast as a protein source in medfly larval diet without any effect on the productivity and the biological quality of reared medfly of VIENNA 8 GSS as assessed by the FAO/IAEA/USDA standard quality control tests. We discuss this finding in the context of mass-rearing and SIT applications.

Combined chemically enhanced primary sedimentation and biofiltration process for low cost municipal wastewater treatment.

The main objective of wastewater treatment is to remove carbon and other nutrients from municipal and industrial effluents in order to protect the environment and human health. Typical wastewater treatment is usually achieved by a combination of physical, chemical and biological methods. In this work, municipal wastewater was depurated using chemically enhanced primary treatment (CEPT) in combination with a pilot-scale trickling filter. Lab scale experiments (Jar-tests) were carried out in order to determine the optimum dosage of chemicals. Selection criteria were the organic load removal efficiency and the low operational cost. Coagulation-flocculation process was conducted through polyaluminium chloride (PAC) and the cationic polyelectrolyte (Zetag 8180) addition. By combining CEPT and trickling filter, tCOD (total Chemical Oxygen Demand), sCOD (soluble Chemical Oxygen Demand), BOD5 (5-day Biochemical Oxygen Demand), NH4+-N, TSS (Total Suspended Solids), VSS (Volatile Suspended Solids) and PO43--P removal efficiencies were estimated to be 89, 82, 93, 60, 96, 96 and 78%, respectively. It is concluded that biological filtration contributed significantly in nutrients removal processes. Moreover, the obtained effluent was low in carbon and rich in nitrogen, which can be applied for restricted irrigation after disinfection, complying with the discharge limits set in the Greek Joint Ministerial Decree 145116/2011.

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.

Characterization of Fungi Associated With Wood Decay of Tree Species and Grapevine in Greece.

A two-year survey was conducted to identify fungi associated with wood decay in a range of tree species and grapevine. Fifty-eight fungal strains isolated from plants of 18 species showing typical wood decay symptoms were characterized by morphological, physiological, and molecular analyses. By 5.8S rRNA gene-ITS sequencing analysis, these isolates were classified into 25 distinct operational taxonomic units, including important phytopathogenic species of the phyla Pezizomycotina and Agaricomycotina, such as Fomitiporia, Inonotus, Phellinus, Inocutis, Fuscoporia, Trametes, Fusarium, Eutypa, Phaeomoniella, Phaeoacremonium, and Pleurostomophora spp. The white rot basidiomycetes Fomitiporia mediterranea (20 isolates, 34.5%) and Inonotus hispidus (6 isolates, 10.3%) were the most prevalent. Pathogenicity tests revealed for the first time that certain fungal species of the genera Fomitiporia, Inonotus, Phellinus, Pleurostomophora, and Fusarium caused wood infection of various tree species in Greece and worldwide. To the best of our knowledge, this is the first report of F. mediterranea as the causal agent of wood decay in pear, pomegranate, kumquat, and silk tree. This is also the first record of Inonotus hispidus, Phellinus pomaceus, Pleurostomophora richardsiae, and Fusarium solani in apple, almond, avocado, and mulberry tree, respectively, whereas P. richardsiae was associated with wood infection of olive tree for the first time in Greece. Cross pathogenicity tests with F. mediterranea strains originated from grapevine applied on other woody hosts and from olive on grapevine demonstrated partial host specificity of the fungus. The potential of F. mediterranea to transinfect hosts other than those originated, along with the host range extension of the fungus, is discussed.

Microbiological and physicochemical evaluation of the effluent quality in a membrane bioreactor system to meet the legislative limits for wastewater reuse.

The aim of this study was to assess the efficacy and effluent quality of a pilot-scale intermittently aerated and fed, externally submerged membrane bioreactor (MBRes) treating municipal wastewater. The effluent quality of the MBRes was evaluated regarding system ability to comply with the Greek legislative limits for restricted and unrestricted wastewater reuse. The average permeate flux was 13.9 L m-2 h-1, while the transmembrane pressure remained above the level of -110 mbar. Experimental data showed that biochemical oxygen demand, chemical oxygen demand, total nitrogen, PO43-- P and total suspended solids removal efficiencies were 97.8, 93.1, 89.6, 93.2 and 100%, respectively, whereas turbidity was reduced by 94.1%. Total coliforms and Escherichia coli were fully eliminated by ultrafiltration and disinfection methods, such as chlorination and ultraviolet radiation. In agreement with the Greek legislation (Joint Ministerial Decree 145116/11) and the guidelines recommended for the Mediterranean countries, the disinfected effluent of the MBRes system can be safely reused directly for urban purposes.

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.

Dominance of rumen microorganisms during cheese whey acidification: acidogenesis can be governed by a rare Selenomonas lacticifex-type fermentation.

The microbial basis of acidification process during spontaneous cheese whey wastewater fermentation was decrypted by implementing both culture-dependent and culture-independent techniques. Lac tobacillus and Bifidobacterium were the predominant taxa among the microbiota growing on MRS (deMan, Rogosa, and Sharpe), while Kazachstania unispora and Dekkera anomala yeast species were also isolated. Almost all Lactobacillus isolates were heterofermentative that could ferment glucose and lactose, with most of them being related to Lactobacillus hilgardii (99.0-100 % similarity). By employing fluorescence techniques, the dominance of long crescent-shaped bacteria in the acidogenic sludge was observed. Temperature gradient gel electrophoresis (TGGE), clone library, and next-generation sequencing techniques revealed the dominance of Selenomonas lacticifex. Based on Illumina data, Selenomonas in the continuous stirred-tank reactor (CSTR) represented 70.13 ± 4.64 % of the bacterial reads, while other Veillonellaceae taxa (Megasphaera and Pectinatus) represented a notable proportion (6.54 %). Prevotella was only detected by Illumina sequencing as an important constituent of the microbial population (14.97 ± 1.71 %). Budding yeasts represented 97 % of the fungal population in the CSTR, with Yarrowia strains representing 88.85 ± 5.52 % of the fungal reads. Spontaneous cheese whey acidification can favor the dominance of rumen bacteria and here was driven by the rarely reported S. lacticifex-type fermentation, which should be taken into consideration during evaluation of acidogenesis in process simulation and modelling. Moreover, the important nervonic acid content detected indicates that acidogenic sludge can be used as a source for the production of high value-added biomedical substrates.

Performance and metabolic aspects of a novel enhanced biological phosphorus removal system with intermittent feeding and alternate aeration.

A novel enhanced biological phosphorus removal (EBPR) system, which combined the intermittent feeding design with an anaerobic selector, was examined using on-line oxidation reduction potential (ORP), nitrate and ammonium probes. Two experimental periods were investigated: the aerobic and anoxic phases were set at 40 and 20 minutes respectively for period I, and set at 30 and 30 minutes for period II. Chemical oxygen demand (COD), biochemical oxygen demand (BOD5) and P removal were measured as high as 87%, 96% and 93% respectively, while total Kjeldahl nitrogen (TKN) and NH4(+) removal averaged 85% and 91%. Two specific denitrification rates (SDNRs), which corresponded to the consumption of the readily biodegradable and slowly biodegradable COD, were determined. SDNR-1 and SDNR-2 during period I were 0.235 and 0.059 g N g(-1) volatile suspended solids (VSS) d(-1) respectively, while the respective rates during period II were 0.105 and 0.042 g N g(-1) VSS d(-1). The specific nitrate formation and ammonium oxidizing rates were 0.076 and 0.064 g N g(-1) VSS d(-1) for period I and 0.065 and 0.081 g N g(-1) VSS d(-1) for period II respectively. The specific P release rates were 2.79 and 4.02 mg P g(-1) VSS h(-1) during period I and II, while the respective anoxic/aerobic uptake rates were 0.42 and 0.55 mg P g(-1) VSS h(-1). This is the first report on an EBPR scheme using the intermittent feeding strategy.

Continuous biohydrogen production from fruit wastewater at low pH conditions.

Biohydrogen production from a simulated fruit wastewater (soluble COD = 3.17 ± 0.10 g L⁻¹) was carried out in a continuous stirred tank reactor (CSTR) of 2 L operational volume without biomass inoculation, heat pre-treatment or pH adjustment, resulting in a low operational pH (3.75 ± 0.09). The hydraulic retention time (HRT) varied from 15 to 5 h. A strong negative correlation (p < 0.01) between the biogas production rate and the HRT was observed. Biogas production rates were higher at 30 °C than at 25 °C (p < 0.01), when the CSTR was operated under the same HRT. The biogas hydrogen content was estimated as high as 55.8 ± 2.3 % and 55.4 ± 2.5 % at 25 and 30 °C, respectively. The main fermentation end products were acetic and butyric acids, followed by ethanol. Significant differences (p < 0.01) during the operation of the CSTR at 25 or 30 °C were identified for butyric acid at almost all HRTs examined. Simulation of the acidogenesis process in the CSTR (based on COD and carbon balances) indicated the possible metabolic compounds produced at 25 and 30 °C reactions and provided an adequate fit of the experimental data.

Intermittent operation of low pressure UF membranes for sewage reuse at household level.

A household-scale wastewater treatment system was operated with domestic sewage. The system could recover gardening/irrigation water from raw sewage or secondary effluent by low pressure ultrafiltration (UF). The UF membranes (surface area = 3.5 m(2), pore size = 0.04 µm) were operated at constant transmembrane pressure (0.13 bar). The proposed technology was examined for approximately 2 months without membrane cleaning. Membrane operation was performed periodically (one or two times per week), simulating water usage for gardening irrigation. During raw sewage filtration (chemical oxygen demand (COD) total = 242 ± 71 mg L(-1), COD soluble = 105 ± 51 mg L(-1), suspended solids = 188 ± 58 mg L(-1)), low permeate COD was achieved (52 ± 25 mg L(-1)), whereas nitrogen and phosphorus were recovered in the permeate. The water recovered during 1 h of operation displayed a gradual decrease from 42 to 22 L m(-2)h(-1) during the 50-d time period. For the secondary effluent filtration, the UF module achieved consistently a recovery rate of 39.6 ± 8.0 L m(-2)h(-1), with an average permeate COD of 37 mg L(-1). In this case, the fouling layer (cake layer) was completely reversible after the relaxation period, rendering the process suitable for unattended household applications.

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.

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.

Molecular characterization of denitrifying bacteria isolated from the anoxic reactor of a modified DEPHANOX plant performing enhanced biological phosphorus removal.

Enhanced Biological Phosphorus Removal (EBPR) under anoxic conditions was achieved using a Biological Nutrient Removal (BNR) system based on a modification of the DEPHANOX configuration. Double-probe Fluorescence in Situ Hybridization (FISH) revealed that Polyphosphate Accumulating Organisms (PAOs) comprised 12.3 +/- 3.2% of the total bacterial population in the modified DEPHANOX plant. The growing bacterial population on blood agar and Casitone Glycerol Yeast Autolysate agar (CGYA) medium was 16.7 +/- 0.9 x 10(5) and 3.0 +/- 0.6 x 10(5) colony forming units (cfu) mL(-1) activated sludge, respectively. A total of 121 bacterial isolates were characterized according to their denitrification ability, with 26 bacterial strains being capable of reducing nitrate to gas. All denitrifying isolates were placed within the alpha-, beta-, and gamma-subdivisions of Proteobacteria and the family Flavobacteriaceae. Furthermore, a novel denitrifying bacterium within the genus Pseudomonas was identified. This is the first report on the isolation and molecular characterization of denitrifying bacteria from EBPR sludge using a DEPHANOX-type plant.

Raw and fungal-treated olive-mill wastewater effects on selected parameters of lettuce (Lactuca sativa L.) growth--the role of proline.

Olive-mill wastewater (OMW) constitutes a major agricultural waste stream for which disposal is associated with significant environmental repercussions. No data are available on the effects of biotreated OMW and of the protective role of exogenously provided proline on plant physiology. In the present study, OMW was administered, either raw or previously treated by the white-rot fungus Pleurotus ostreatus, with or without proline amendment, to lettuce plants growing in sterilized sand. Biotreated OMW and proline addition resulted in significant moderation of OMW adverse effects on plant biomass production and ascorbic acid content, while their synergistic action alleviated the severe negative impact on net photosynthetic rate, water use efficiency and photosynthetic activity (Fv/Fo) invoked by the effluent. Moreover, biotreated OMW supplemented with proline, moderated the decrease in chlorophylls exerted by raw OMW, but it did not contribute at restoring carotenoids content. Restoration of plant transpiration was complete when biotreated OMW was used (with or without proline); proline alone mitigated the negative impact of OMW on photosynthetic efficiency (Fv/Fm and Fv'/Fm'). It seems that key photosynthetic parameters could be exploited as suitable evaluators of wastewater-induced plant toxicity, while plant fertigation with biotreated and/or supplemented OMW could be an interesting prospect in valorizing this effluent.

Bioprocess performance, transformation pathway, and bacterial community dynamics in an immobilized cell bioreactor treating fludioxonil-contaminated wastewater under microaerophilic conditions.

Fludioxonil is a post-harvest fungicide contained in effluents produced by fruit packaging plants, which should be treated prior to environmental dispersal. We developed and evaluated an immobilized cell bioreactor, operating under microaerophilic conditions and gradually reduced hydraulic retention times (HRTs) from 10 to 3.9 days, for the biotreatment of fludioxonil-rich wastewater. Fludioxonil removal efficiency was consistently above 96%, even at the shortest HRT applied. A total of 12 transformation products were tentatively identified during fludioxonil degradation by using liquid chromatography coupled to quadrupole time-of-flight Mass spectrometry (LC-QTOF-MS). Fludioxonil degradation pathway was initiated by successive hydroxylation and carbonylation of the pyrrole moiety and disruption of the oxidized cyanopyrrole ring at the NH-C bond. The detection of 2,2-difluoro-2H-1,3-benzodioxole-4-carboxylic acid verified the decyanation and deamination of the molecule, whereas its conversion to the tentatively identified compound 2,3-dihydroxybenzoic acid indicated its defluorination. High-throughput amplicon sequencing revealed that HRT shortening led to reduced α-diversity, significant changes in the ß-diversity, and a shift in the bacterial community composition from an initial activated sludge system typical community to a community composed of bacterial taxa like Clostridium, Oligotropha, Pseudomonas, and Terrimonas capable of performing advanced degradation and/or aerobic denitrification. Overall, the immobilized cell bioreactor operation under microaerophilic conditions, which minimizes the cost for aeration, can provide a sustainable solution for the depuration of fludioxonil-contaminated agro-industrial effluents.

Isolation, characterization and industrial application of a Cladosporium herbarum fungal strain able to degrade the fungicide imazalil.

Imazalil (IMZ) is an imidazole fungicide commonly used by fruit-packaging plants (FPPs) to control fungal infections during storage. Its application leads to the production of pesticide-contaminated wastewaters, which, according to the European Commission, need to be treated on site. Considering the lack of efficient treatment methods, biodepuration systems inoculated with tailored-made inocula specialized on the removal of such persistent fungicides appear as an appropriate solution. However, nothing is known about the biodegradation of IMZ. We aimed to isolate and characterize microorganisms able to degrade the recalcitrant fungicide IMZ and eventually to test their removal efficiency under near practical bioengineering conditions. Enrichment cultures from a soil receiving regular discharges of effluents from a FPP, led to the isolation of a Cladosporium herbarum strain, which showed no pathogenicity on fruits, a trait essential for its biotechnological exploitation in FPPs. The fungus was able to degrade up to 100 mg L-1 of IMZ. However, its degrading capacity and growth was reduced at increasing IMZ concentrations in a dose-dependent manner, suggesting the involvement of a detoxification rather than an energy-gain mechanism in the dissipation of IMZ. The isolate could tolerate and gradually degrade the fungicides fludioxonil (FLD) and thiabendazole (TBZ), also used in FPPs and expected to coincide alongside IMZ in FPP effluents. The capacity of the isolate to remove IMZ in a practical context was evaluated in a benchtop immobilized-cell bioreactor fed with artificial IMZ-contaminated wastewater (200 mg L-1). The fungal strain established in the reactor, completely dominated the fungal community and effectively removed >96% of IMZ. The bioreactor also supported a diverse bacterial community composed of Sphingomonadales, Burkholderiales and Pseudomonadales. Our study reports the isolation of the first IMZ-degrading microorganism with high efficiency to remove IMZ from agro-industrial effluents under bioengineering conditions.

Algae and Their Metabolites as Potential Bio-Pesticides.

An increasing human population necessitates more food production, yet current techniques in agriculture, such as chemical pesticide use, have negative impacts on the ecosystems and strong public opposition. Alternatives to synthetic pesticides should be safe for humans, the environment, and be sustainable. Extremely diverse ecological niches and millions of years of competition have shaped the genomes of algae to produce a myriad of substances that may serve humans in various biotechnological areas. Among the thousands of described algal species, only a small number have been investigated for valuable metabolites, yet these revealed the potential of algal metabolites as bio-pesticides. This review focuses on macroalgae and microalgae (including cyanobacteria) and their extracts or purified compounds, that have proven to be effective antibacterial, antiviral, antifungal, nematocides, insecticides, herbicides, and plant growth stimulants. Moreover, the mechanisms of action of the majority of these metabolites against plant pests are thoroughly discussed. The available information demonstrated herbicidal activities via inhibition of photosynthesis, antimicrobial activities via induction of plant defense responses, inhibition of quorum sensing and blocking virus entry, and insecticidal activities via neurotoxicity. The discovery of antimetabolites also seems to hold great potential as one recent example showed antimicrobial and herbicidal properties. Algae, especially microalgae, represent a vast untapped resource for discovering novel and safe biopesticide compounds.