Inhibition of the respiratory chain reactions in denitrifying EBPR biomass under simultaneous presence of acetate and electron acceptor.

In this study, the deterioration of the typical EBPR (Enhanced Biological Phosphorus Removal) process due to the simultaneous presence of electron donor (external substrate) and electron acceptor (oxygen or nitrate) was investigated by using a PAOs (Polyphosphate Accumulating Organisms)-enriched biomass grown in a modified DEPHANOX system. Intracellular and extracellular constituents were monitored in batch tests under different electron donor and acceptor conditions and specific oxygen and nitrogen uptake rates were evaluated. Results showed that phosphorus uptake was inhibited during the simultaneous presence of electron donor (acetate) and acceptor (O2/NO3-) in the mixed liquor. In the presence of acetate, PHAs and glycogen were produced under both aerobic and anoxic conditions irrespectively to the PHAs amount already stored intracellularly. The Krebs cycle reactions and oxidative phosphorylation provided the reduced coenzymes and energy required for PHAs synthesis when biomass polyphosphate content was low. On the contrary, polyphosphate cleavage provided the ATP required for PHAs synthesis in the presence of high biomass polyphosphate content. Inhibition of the respiratory chain reactions was observed when biomass with high polyphosphate and low PHAs content was subjected to simultaneous presence of electron donor and acceptor. PHAs utilization rather than glycogen degradation appears to favor phosphate accumulation since no polyphosphate synthesis occurred in the absence of PHAs reserves.

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.

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.

Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus.

Thirty-nine white-rot fungi belonging to nine species of Agaricomycotina (Basidiomycota) were initially screened for their ability to decrease olive-mill wastewater (OMW) phenolics. Four strains of Ganoderma australe, Ganoderma carnosum, Pleurotus eryngii and Pleurotus ostreatus, were selected and further examined for key-aspects of the OMW biodegradation process. Fungal growth in OMW-containing batch cultures resulted in significant decolorization (by 40-46% and 60-65% for Ganoderma and Pleurotus spp. respectively) and reduction of phenolics (by 64-67% and 74-81% for Ganoderma and Pleurotus spp. respectively). COD decrease was less pronounced (12-29%). Cress-seeds germination increased by 30-40% when OMW was treated by Pleurotus strains. Toxicity expressed as inhibition of Aliivibrio fischeri luminescence was reduced in fungal-treated OMW samples by approximately 5-15 times compared to the control. As regards the pertinent enzyme activities, laccase and Mn-independent peroxidase were detected for Ganoderma spp. during the entire incubation period. In contrast, Pleurotus spp. did not exhibit any enzyme activities at early growth stages; instead, high laccase (five times greater than those of Ganoderma spp.) and Mn peroxidases activities were determined at the end of treatment. OMW decolorization by Ganoderma strains was strongly correlated to the reduction of phenolics, whereas P. eryngii laccase activity was correlated with the effluent's decolorization.

Denitrifying polyphosphate accumulating organisms population and nitrite reductase gene diversity shift in a DEPHANOX-type activated sludge system fed with municipal wastewater.

Enhanced biological phosphorus removal (EBPR) is a widely applied method for nutrients removal, although little is known about the key genes regulating the complex biochemical transformations occurring in activated sludge during phosphorus removal. In the present study, the nitrite reductase gene (nirS) diversity and the denitrifying polyphosphate accumulating organisms (DPAOs) population, grown in a bench scale, two-sludge, continuous flow plant, operating for biological anoxic phosphorus removal (DEPHANOX-type), fed with municipal wastewater, were examined by means of physicochemical analyses and the application of molecular techniques. The DEPHANOX configuration highly influenced biomass phosphorus as well as polyhydroxyalkanoates content and facilitated the enrichment of the DPAOs population. The application of double probe fluorescent in situ hybridization (double probe FISH) technique revealed that DPAOs comprised 20% of the total bacterial population. Based on clone libraries construction and nirS gene sequencing analysis, a pronounced shift in denitrifying bacteria diversity was identified during activated sludge acclimatization. Moreover, nirS gene sequences distinct from those detected in any known bacterial strain or environmental clone were identified. This is the first report studying the microbial properties of activated sludge in a DEPHANOX-type system using molecular techniques.

Olivibacter sitiensis gen. nov., sp. nov., isolated from alkaline olive-oil mill wastes in the region of Sitia, Crete.

A novel, Gram-negative, non-motile, non-sporulating, rod-shaped bacterium isolated from a viscous two-phase olive-oil mill waste ('alpeorujo') is described. The strain, designated AW-6T, is an obligate aerobe, forming irregular, pigmented creamy white colonies. The pH and temperature ranges for growth were pH 5-8 and 5-45 degrees C, with optimal pH and temperature for growth of pH 6-7 and 28-32 degrees C, respectively. Strain AW-6T was chemo-organotrophic and utilized mostly D+ -glucose, protocatechuate and D+ -xylose, followed by L-cysteine, D- -fructose, D+ -galactose, L-histidine, lactose, sorbitol and sucrose. Menaquinone-7 was detected in the respiratory chain of strain AW-6T. The major fatty acids of strain AW-6T were C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH, iso-C(15 : 0), iso-C(17 : 0) 3-OH and C(16 : 0). The closest phylogenetic relative of strain AW-6T was clone BIti35 (89.7 % 16S rRNA gene sequence similarity), while Sphingobacterium thalpophilum DSM 11723T was the closest recognized relative within the Sphingobacteriaceae (88.2 % similarity). Strain AW-6T showed a low level of DNA-DNA relatedness to S. thalpophilum DSM 11723T (33.8-37.0 %). The DNA G+C content of strain AW-6T was 45.6 mol%. Physiological and chemotaxonomic data further confirmed the distinctiveness of strain AW-6T from members of the genera Sphingobacterium and Pedobacter. Thus, strain AW-6T is considered to represent a novel species of a new genus within the family Sphingobacteriaceae, for which the name Olivibacter sitiensis gen. nov., sp. nov. is proposed. The type strain is AW-6T =DSM 17696T=CECT 7133T).

Ecophysiology and molecular phylogeny of bacteria isolated from alkaline two-phase olive mill wastes.

The use of two-phase centrifugal decanters has been widely adopted in the olive oil extraction industry in order to reduce the huge quantities of wastewaters produced during the traditional three-phase extraction process. The resulting sludge-like byproduct, widely known as "alpeorujo", has a pH of 4-6, low water activity (a(w)) and high phytotoxicity. Addition of Ca(OH)(2) to alpeorujo, which is commonly performed at the olive oil mill to handle disposal problems related to acidic pH and odor emissions, creates an alkaline secondary waste (alkaline alpeorujo). Bacteria isolated from alkaline alpeorujo were cultured in order to investigate their physiological and phylogenetic characteristics. The bacterial population at neutral pH was estimated to be 6.0+/-0.4 x 10(7) cells g(-1) dw, while the bacterial population at pH 11 reached 2.1+/-0.3 x 10(5) cells g(-1) dw. Fourteen strains isolated from alkaline pH were halotolerant alkaliphiles, while seven isolates from neutral pH were moderate to extreme halotolerant or/and alkalitolerant bacteria. Based on 16S rRNA gene sequence analysis, four of the halotolerant alkaliphilic isolates showed 98.4-99.2% similarity to known sequences of Bacillus alcalophilus and Nesterenkonia lacusekhoensis, whereas ten isolates demonstrated low percentage similarities (94.4-96.9%) to the genera Idiomarina, Halomonas and Nesterenkonia. As concerns bacteria isolated from neutral pH, four isolates were associated with Corynebacterium, Novosphingobium, Serratia marcescens and Pseudomonas aeruginosa (98.3-99.9% similarities), while three isolates presented 96.5-97.2% sequence similarities to Rhodobacter, Pseudomonas and Ochrobactrum. At least six groups of isolates represent novel phylogenetic linkages among Bacteria.