Dominant Candidatus Accumulibacter phosphatis Enriched in Response to Phosphate Concentrations in EBPR Process.

Candidatus Accumulibacter phosphatis (Accumulibacter), which plays an important role in enhanced biological phosphorus removal in wastewater treatment plants, is phylogenetically classified into two major types (Types I and II). Phosphate concentrations affect the Accumulibacter community of the biomass enriched in treatment plants. Therefore, in the present study, Accumulibacter enrichments were conducted using a down-flow hanging sponge reactor under five conditions and a wide range of controlled phosphate concentrations in order to investigate how phosphate governs the community. We found that excessive phosphate levels inhibited Accumulibacter activity, that this inhibitory effect was greater for Type II. In addition, the affinity of Type II for phosphate was higher than that of Type I. Type IIA-B dominated at a phosphate concentration less than 5 mg P L-1, while Type IA was dominant at 50 and 500 mg P L-1. These patterns of enrichment may be explained by an inhibition kinetics model.

Influence of pH, EDTA/Fe(II) ratio, and microbial culture on Fe(II)-mediated autotrophic denitrification.

Fe(II)-mediated autotrophic denitrification with four different microbial cultures under different pH and EDTA/Fe(II) conditions was investigated in batch bioassays. Initially, the highest nitrate removal (72%) was achieved with an activated sludge inoculum. The use of pure cultures of Pseudogulbenkiania strain 2002 and Thiobacillus denitrificans resulted in a 55 and 52% nitrate removal, respectively. No denitrification was observed for a mixed culture dominated by Thiobacillus thioparus and T. denitrificans. A longer enrichment on Fe(II) and the supplementation of thiosulfate as additional electron donor were needed to stimulate the denitrifying activity of the Thiobacillus-mixed culture. A second subculture on Fe(II) as sole electron donor resulted in higher denitrification efficiencies for all microbial cultures. In particular, nitrate removal reached up to 84% with a specific nitrate removal rate of 1.160 mM·(g VSS·day)-1 in the bioassays seeded with the Thiobacillus-mixed culture. All cultures were favored by decreasing the EDTA/Fe(II) molar ratio from 2.0 to 0.5. The most significant denitrification enhancement was observed for the Pseudogulbenkiania species, indicating a lower tolerance to EDTA. The two pure cultures effectively maintained denitrification at pH 7.0 and were more sensitive to a pH decrease. Conversely, the optimal pH was 6.0 for the Thiobacillus-mixed and activated sludge cultures.

Bacterial Compatibility in Combined Inoculations Enhances the Growth of Potato Seedlings.

The compatibility of strains is crucial for formulating bioinoculants that promote plant growth. We herein assessed the compatibility of four potential bioinoculants isolated from potato roots and tubers (Sphingomonas sp. T168, Streptomyces sp. R170, Streptomyces sp. R181, and Methylibium sp. R182) that were co-inoculated in order to improve plant growth. We screened these strains using biochemical tests, and the results obtained showed that R170 had the highest potential as a bioinoculant, as indicated by its significant ability to produce plant growth-promoting substances, its higher tolerance against NaCl (2%) and AlCl3 (0.01%), and growth in a wider range of pH values (5.0-10.0) than the other three strains. Therefore, the compatibility of R170 with other strains was tested in combined inoculations, and the results showed that the co-inoculation of R170 with T168 or R182 synergistically increased plant weight over un-inoculated controls, indicating the compatibility of strains based on the increased production of plant growth promoters such as indole-3-acetic acid (IAA) and siderophores as well as co-localization on roots. However, a parallel test using strain R181, which is the same Streptomyces genus as R170, showed incompatibility with T168 and R182, as revealed by weaker plant growth promotion and a lack of co-localization. Collectively, our results suggest that compatibility among bacterial inoculants is important for efficient plant growth promotion, and that R170 has potential as a useful bioinoculant, particularly in combined inoculations that contain compatible bacteria.

Microbial selection on enhanced biological phosphorus removal systems fed exclusively with glucose.

The microbial selection on an enhanced biological phosphorus removal (EBPR) system was investigated in a laboratory-scale sequencing batch reactor fed exclusively with glucose as the carbon source. Fluorescence In Situ Hybridization analysis was performed to target two polyphosphate accumulating organisms (PAOs) (i.e., Candidatus Accumulibacter phosphatis and Microlunatus phosphovorus) and two glycogen accumulating organisms (GAOs) (i.e., Candidatus Competibacter phosphatis and Micropruina glycogenica). The results show that glucose might not select for Candidatus Accumulibacter phosphatis. However, Microlunatus phosphovorus, Candidatus Competibacter phosphatis, and Micropruina glycogenica might be selected. The highest percent relative abundance (% RA) of Candidatus Accumulibacter phosphatis was about 42%; this occurred at the beginning of the experimental period when phosphorus removal was efficient. However, the % RA of these bacteria decreased, reaching below 4% at the end of the run. The maximum % RA of Microlunatus phosphovorus, Candidatus Competibacter phosphatis, and Micropruina glycogenica was about 21, 37, 17%, respectively. It appears that a higher glucose concentration might be detrimental for Microlunatus phosphovorus and Micropruina glycogenica. Results also indicate a dominance of GAOs over PAOs when EBPR systems are fed with glucose. It is possible that the GAOs outcompete the PAOs at low pH values; it has been reported that at low pH, GAOs use glycogen as the energy source to uptake glucose. As a result, P-removal deteriorated. Therefore, glucose is not a strong candidate as a carbon source to supplement EBPR systems that do not contain sufficient volatile fatty acids.

Comparison of microbial community compositions of injection and production well samples in a long-term water-flooded petroleum reservoir.

Water flooding plays an important role in recovering oil from depleted petroleum reservoirs. Exactly how the microbial communities of production wells are affected by microorganisms introduced with injected water has previously not been adequately studied. Using denaturing gradient gel electrophoresis (DGGE) approach and 16S rRNA gene clone library analysis, the comparison of microbial communities is carried out between one injection water and two production waters collected from a working block of the water-flooded Gudao petroleum reservoir located in the Yellow River Delta. DGGE fingerprints showed that the similarities of the bacterial communities between the injection water and production waters were lower than between the two production waters. It was also observed that the archaeal composition among these three samples showed no significant difference. Analysis of the 16S rRNA gene clone libraries showed that the dominant groups within the injection water were Betaproteobacteria, Gammaproteobacteria and Methanomicrobia, while the dominant groups in the production waters were Gammaproteobacteria and Methanobacteria. Only 2 out of 54 bacterial operational taxonomic units (OTUs) and 5 out of 17 archaeal OTUs in the injection water were detected in the production waters, indicating that most of the microorganisms introduced by the injection water may not survive to be detected in the production waters. Additionally, there were 55.6% and 82.6% unique OTUs in the two production waters respectively, suggesting that each production well has its specific microbial composition, despite both wells being flooded with the same injection water.

The influence of biofilms on the migration of uranium in acid mine drainage (AMD) waters.

The uranium mine in Königstein (Germany) is currently in the process of being flooded. Huge mass of Ferrovum myxofaciens dominated biofilms are growing in the acid mine drainage (AMD) water as macroscopic streamers and as stalactite-like snottites hanging from the ceiling of the galleries. Microsensor measurements were performed in the AMD water as well as in the biofilms from the drainage channel on-site and in the laboratory. The analytical data of the AMD water was used for the thermodynamic calculation of the predominance fields of the aquatic uranium sulfate (UO(2)SO(4)) and UO(2)(++) speciation as well as of the solid uranium species Uranophane [Ca(UO(2))(2)(SiO(3)OH)(2)∙5H(2)O] and Coffinite [U(SiO(4))(1-x)(OH)(4x)], which are defined in the stability field of pH>4.8 and Eh<960 mV and pH>0 and Eh<300 mV, respectively. The plotting of the measured redox potential and pH of the AMD water and the biofilm into the calculated pH-Eh diagram showed that an aqueous uranium(VI) sulfate complex exists under the ambient conditions. According to thermodynamic calculations a retention of uranium from the AMD water by forming solid uranium(VI) or uranium(IV) species will be inhibited until the pH will increase to >4.8. Even analysis by Energy-filtered Transmission Electron Microscopy (EF-TEM) and electron energy loss spectroscopy (EELS) within the biofilms did not provide any microscopic or spectroscopic evidence for the presence of uranium immobilization. In laboratory experiments the first phase of the flooding process was simulated by increasing the pH of the AMD water. The results of the experiments indicated that the F. myxofaciens dominated biofilms may have a substantial impact on the migration of uranium. The AMD water remained acid although it was permanently neutralized with the consequence that the retention of uranium from the aqueous solution by the formation of solid uranium species will be inhibited.

Municipal wastewater treatment and biomass accumulation with a wastewater-born and settleable algal-bacterial culture.

A wastewater-born and settleable algal-bacterial culture, cultivated in a stirred tank photobioreactor under lab conditions, was used to remove the carbon and nutrients in municipal wastewater and accumulate biomass simultaneously. The algal-bacterial culture showed good settleable property and could totally settle down over 20 min, resulting in a reduction of total suspended solids from an initial 1.84 to 0.016 g/l. The average removal efficiencies of chemical oxygen demand, total kjeldahl nitrogen and phosphate were 98.2 ± 1.3%, 88.3 ± 1.6% and 64.8 ± 1.0% within 8 days, respectively, while the average biomass productivity was 10.9 ± 1.1 g/m(2) · d. Accumulation into biomass, identified as the main nitrogen and phosphorus removal mechanism, accounted for 44.9 ± 0.4% and 61.6 ± 0.5% of total inlet nitrogen and phosphorus, respectively. Microscopic analysis showed the main algae species in the bioreactor were filamentous blue-green algae. Furthermore, denaturing gradient gel electrophoresis and 16S rDNA gene sequencing revealed that the main bacteria present in the photobioreactor were consortia with sequences similar to those of Flavobacteria, Gammaproteobacteria, Bacteroidia and Betaproteobacteria. This study explores a better understanding of an algae-bacteria system and offers new information on further usage of biomass accumulated during treatment.

Effect of pH reduction on polyphosphate- and glycogen-accumulating organisms in enhanced biological phosphorus removal processes.

We investigated the effect of pH reduction on polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) in the enhanced biological phosphorus removal (EBPR) process. Three laboratory-scale EBPR reactors were used. Initially, the reactors were operated at pH 7.9±0.1 and 6.5±0.1, and after 27 days, the pH was lowered to 6.5±0.1 and 6.0±0.1, respectively. PAOs and GAOs were monitored using real-time quantitative polymerase chain reaction and/or fluorescent in situ hybridization. Phosphorus removal performance was also monitored. During the start-up period, high EBPR activity and increases in Candidatus 'Accumulibacter phosphatis' (Accumulibacter) and Candidatus 'Competibacter phosphatis' (Competibacter) were observed. In all runs, Accumulibacter and Competibacter were the dominant PAO and GAO, respectively. Accumulibacter began to decline 10-18 days after lowering the pH to 6.5±0.1. After lowering the pH to 6.0±0.1, the Accumulibacter population decreased immediately. Contrastingly, an obvious adverse effect of pH reduction on Competibacter was not observed. In all runs, EBPR activity began to deteriorate 6-12 days after Accumulibacter decline began. Thus, our results show that pH reduction had an immediate or delayed effect on Accumulibacter decline. Moreover, the time lag between the start of Accumulibacter decline and that of EBPR deterioration implies that EBPR deterioration by pH reduction went through unknown process.

Modelling the population dynamics and metabolic diversity of organisms relevant in anaerobic/anoxic/aerobic enhanced biological phosphorus removal processes.

In this study, enhanced biological phosphorus removal (EBPR) metabolic models are expanded in order to incorporate the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) under sequential anaerobic/anoxic/aerobic conditions, which are representative of most full-scale EBPR plants. Since PAOs and GAOs display different denitrification tendencies, which is dependent on the phylogenetic identity of the organism, the model was separated into six distinct biomass groups, constituting Accumulibacter Types I and II, as well as denitrifying and non-denitrifying Competibacter and Defluviicoccus GAOs. Denitrification was modelled as a multi-step process, with nitrate (NO(3)), nitrite (NO(2)), nitrous oxide (N(2)O) and di-nitrogen gas (N(2)) being the primary components. The model was calibrated and validated using literature data from enriched cultures of PAOs and GAOs, obtaining a good description of the observed biochemical transformations. A strong correlation was observed between Accumulibacter Types I and II, and nitrate-reducing and non-nitrate-reducing PAOs, respectively, where the abundance of each PAO subgroup was well predicted by the model during an acclimatization period from anaerobic-aerobic to anaerobic-anoxic conditions. Interestingly, a strong interdependency was observed between the anaerobic, anoxic and aerobic kinetic parameters of PAOs and GAOs. This could be exploited when metabolic models are calibrated, since all of these parameters should be changed by an identical factor from their default value. Factors that influence these kinetic parameters include the fraction of active biomass, relative aerobic/anoxic fraction and the ratio of acetyl-CoA to propionyl-CoA. Employing a metabolic approach was found to be advantageous in describing the performance and population dynamics in such complex microbial ecosystems.

Metatranscriptomic array analysis of 'Candidatus Accumulibacter phosphatis'-enriched enhanced biological phosphorus removal sludge.

Here we report the first metatranscriptomic analysis of gene expression and regulation of 'Candidatus Accumulibacter'-enriched lab-scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. RNA samples were collected at the early stage of anaerobic and aerobic phases (15 min after acetate addition and switching to aeration respectively). We detected the expression of a number of genes involved in the carbon and phosphate metabolisms, as proposed by EBPR models (e.g. polyhydroxyalkanoate synthesis, a split TCA cycle through methylmalonyl-CoA pathway, and polyphosphate formation), as well as novel genes discovered through metagenomic analysis. The comparison between the early stage anaerobic and aerobic gene expression profiles showed that expression levels of most genes were not significantly different between the two stages. The majority of upregulated genes in the aerobic sample are predicted to encode functions such as transcription, translation and protein translocation, reflecting the rapid growth phase of Accumulibacter shortly after being switched to aerobic conditions. Components of the TCA cycle and machinery involved in ATP synthesis were also upregulated during the early aerobic phase. These findings support the predictions of EBPR metabolic models that the oxidation of intracellularly stored carbon polymers through the TCA cycle provides ATP for cell growth when oxygen becomes available. Nitrous oxide reductase was among the very few Accumulibacter genes upregulated in the anaerobic sample, suggesting that its expression is likely induced by the deprivation of oxygen.

Biomass granulation in an aerobic:anaerobic-enhanced biological phosphorus removal process in a sequencing batch reactor with varying pH.

Long-term influences of different steady-state pH conditions on microbial community composition were determined by fluorescence in situ hybridization (FISH) in a laboratory scale reactor configured for enhanced biological phosphorus removal (EBPR). Chemical profiles were consistent with shifts in populations from polyphosphate-accumulating organisms (PAO) to glycogen-accumulating organisms (GAO) when pH fell from pH 7.5 to 7.0 and then to 6.5. While biomass was both dispersed and flocculated at pH 7.5, almost complete granulation occurred gradually after pH was dropped to 7.0, and these granules increased in size as the pH was reduced further to 6.5. Reverting back to pH 7.5 led to granule breakdown and corresponding increases in anaerobic phosphate release. Granules consisted almost entirely of Accumulibacter PAO cells, while putative GAO populations were always present in small numbers. Results suggest that low pH may contribute to granulation under these operational conditions. While chemical profiles suggested the PAO:GAO balance was changing as pH fell, FISH failed to reveal any marked corresponding increase in GAO abundances. Instead, TEM evidence suggested the Accumulibacter PAO phenotype was becoming more like that of a GAO. These data show how metabolically adaptable the Accumulibacter PAO can be under anaerobic:aerobic conditions in being able to cope with marked changes in plant conditions. They suggest that decreases in EBPR capacity may not necessarily reflect shifts in community composition, but in the existing population metabolism.

The microbiology of phosphorus removal in activated sludge processes-the current state of play.

This review discusses critically what we know and would like to know about the microbiology of phosphorus (P) removal in activated sludge systems. In particular, the description of the genome sequences of two strains of the polyphosphate accumulating organism found in these processes, Candidatus 'Accumulibacter phosphatis', allows us to address many of the previously unanswered questions relating to how these processes behave, and to raise new questions about the microbiology of P removal. This article attempts to be deliberately speculative, and inevitably subjective, but hopefully at the same time useful to those who have an active interest in these environmentally very important processes.

Microbial distribution of Accumulibacter spp. and Competibacter spp. in aerobic granules from a lab-scale biological nutrient removal system.

Granular sludge for simultaneous nitrification, denitrification and phosphorus removal (SNDPR) was generated and studied in a lab-scale sequencing batch reactor (SBR). The SBR was monitored for 450 days during which the biomass was transformed from flocs to granules, which persisted for the last 130 days of operation. Short sludge settling time was employed to successfully generate the granules, with the 10th and 90th percentiles of diameter being 0.7 and 1.6 mm respectively. Good phosphorus removal and nitrification occurred throughout the SBR operation but only when granules were generated were denitrification and full nutrient removal complete. Fluorescence in situ hybridization and oxygen microsensors were used to study the granules at a microscale. Accumulibacter spp. (a polyphosphate-accumulating organism, PAO) and Competibacter spp. (a glycogen non-polyphosphate-accumulating organism, GAO) were the most abundant microbial community members (together 74% of all Bacteria) and both are capable of denitrification. In the aerobic period of the SBR operation, the oxygen penetrated 250 microm into the granules leaving large anoxic zones in the centre part where denitrification can occur. In granules > 500 microm in diameter, Accumulibacter spp. was dominant in the outermost 200 microm region of the granule while Competibacter spp. dominated in the granule central zone. The stratification of these two populations between the outer aerobic and inner anoxic part of the granule was highly significant (P < 0.003). We concluded that the GAO Competibacter spp., and not the PAO Accumulibacter spp., was responsible for denitrification in this SBR. This is undesirable for SNDPR as savings in carbon demand cannot be fulfilled with phosphorus removal and denitrification being achieved by different groups of bacteria.

Significant changes in the bacterioplankton community structure of a maritime Antarctic freshwater lake following nutrient enrichment.

Nutrient enrichment is known to increase bacterioplankton population density in a variety of Antarctic freshwater lakes. However, relatively little is known about the associated changes in species composition. In this study, the bacterioplankton community composition of one such lake was studied following natural nutrient enrichment to investigate the resistance of the system to environmental change. Heywood Lake is an enriched freshwater maritime Antarctic lake, with nitrogen and phosphorus concentrations significantly higher than its more oligotrophic neighbours (by at least an order of magnitude). This major change in lake chemistry has occurred following large increases in the fur seal population over the last 30 years. Using analysis of 16S rRNA gene fragments, fatty acid methyl ester analysis, denaturing gradient gel electrophoresis and fluorescence in situ hybridization, significant changes are reported in lake microbiology which have resulted in a distinct bacterioplankton community. In comparison to its more oligotrophic neighbours, nutrient-enriched Heywood Lake has a high bacterioplankton population density, reduced species richness and an increasing evenness among key groups. Only 42.3 % of the clones found with > or =97 % similarity to a named genus were also present in adjacent oligotrophic lakes, including three of the dominant groups. Critically, there was an apparent shift in dominance with trophic status (from the beta-Proteobacteria to the Actinobacteria). Other key observations included the absence of a dominant group of Cyanobacteria and the presence of marine bacteria. The significant impact of natural nutrient enrichment on the microbiology of Heywood Lake, therefore, suggests that low-temperature oligotrophic freshwater lake systems might have low resistance to environmental change.

Changes in phosphorus removing performance and bacterial community structure in an enhanced biological phosphorus removal reactor.

A lab-scale-enhanced biological phosphorus removal (EBPR) reactor was operated for 204 days to investigate the correlation between phosphorus removing performance and bacterial community structure. The phosphorus removing performance was good from day 1 to 92 and from day 172 to 204. However, the removal activity was in a deteriorated state from day 93 to 171. From day 69 (2 weeks before the beginning of the deterioration) to 118 (2 weeks after the beginning of the deterioration), sludge P content decreased. The amounts of ubiquinone-8 and menaquinone-8 (H(4)) decreased during this period while the amount of ubiquinone-10 increased. The comparison of these changes and the general attribution of each quinone to the bacterial phylogenetic groups suggested that beta proteobacteria and Actinobacteria contributed to EBPR positively, and that alpha proteobacteria were related to this EBPR deterioration. Glycogen accumulating organisms (GAOs) are considered to detrimentally affect EBPR ability by outcompeting the phosphorus accumulating organisms by using aerobically synthesized glycogen as the energy source to assimilate organic substrates anaerobically to form polyhydroxyalkanoates. However, in this research, there was nearly no substrate uptake during the anaerobic period at the middle of the deteriorated performance period. This suggests that the deterioration observed in this research does not agree with the GAOs inhibition model. In this research, the excess P release at the anaerobic period was concluded to cause the deterioration.

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants.

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called 'Candidatus Accumulibacter phosphatis' was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge 'biography' be well understood--i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked.

Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato.

AIMS: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death. METHODS AND RESULTS: It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine-killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR-amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR-amplification resulted in positive reactions also long after cells were dead. The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts. CONCLUSIONS: NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.

Selective enrichment and characterization of a phosphorus-removing bacterial consortium from activated sludge.

Under alternating aerobic/anaerobic conditions and without additional carbon sources, a bacterial consortium consisting initially of 18 bacterial strains was obtained in a sequence batch reactor. The phosphorus removal capability could only be maintained using sterile filtrate of activated sludge as medium. The addition of calcium and magnesium salts, as well as vitamins and trace elements, to autoclaved sterile filtrate of activated sludge was not sufficient to achieve stable phosphorus removal. A further enrichment by subcultivation on solid, agar, freezing, and shortening of the aerobic and anaerobic phases led to a defined bacterial consortium consisting of four strains. On the basis of physiological and chemotaxonomic characterization, and partial 16S rRNA sequencing, one of the organisms was identified as Delftia acidovorans. A further isolate belonged to the Bacillus cereus group, and the third isolate was identified as Microbacterium sp.. The remaining strain seems to represent a new genus within the Flavobacteriaceae. Under continuous chemostat conditions, this consortium was able to remove up to 9.6 mg P/l phosphate in the aerobic phase and released up to 8.5 mg/l in the anaerobic phase. Up to 25 mg P-polyphosphate/g dry mass was stored under aerobic conditions.

Biodegradation of hydroxylated and methoxylated benzoic, phenylacetic and phenylpropenoic acids present in olive mill wastewaters by two bacterial strains.

Two aerobic bacterial strains, a chlorophenol-degrading bacterium characterized in this work as a Ralstonia sp. LD35 on the basis of the sequence of the gene encoding for 16S ribosomal RNA, and Pseudomonas putida DSM 1868, capable of metabolizing 4-methoxybenzoic acid, were tested for their capacity to degrade monocyclic aromatic acids responsible for the toxicity of olive mill wastewaters (OMWs). Both strains possess interesting and complementary degradation capabilities in resting cell conditions: Ralstonia sp. LD35 was found to metabolize 4-hydroxybenzoic, 4-hydroxyphenylacetic, 3,4-dihydroxycinnamic and cinnamic acid, whereas DSM 1868 was capable of metabolizing 4-hydroxy-3-methoxybenzoic, 3,4-dimethoxybenzoic and 4-hydroxy-3,5-dimethoxybenzoic acid, as well as 4-hydroxybenzoic and 4-hydroxyphenylacetic acid. The kinetic parameters describing the growth of the two strains on the same compounds were determined in growing-cell batch conditions, and showed that both strains presented high affinity and high specific growth rates towards all assayed substrates. In addition, the two strains were capable of growing on and extensively biodegrading a mixture of monocyclic aromatic acids commonly found at high concentrations in OMWs, and of growing on a 20% dilution of a natural OMW. All these features make the two strains attractive candidates for the development of a biotechnological process for the biodegradation of aromatic compounds found in OMWs.