The influence of storage on the morphology and physiology of phthalic acid-degrading aerobic granules.

The precultured aerobic granules with special degradabilities could be used as a feasible bioseed for enhancement of aerobic granulation systems. In practice, the storage stability, physicochemical characteristics, and recovering efficiency of granules are crucial for a long-distance transportation and successful application. In this study, phthalic acid (PA)-degrading aerobic granules were cultivated and stored for 8 wk at 4 degrees C. The granular size, settling ability as well as structure integrity was found stable during the storage period. It was observed that the upper 1/3 part of granules stored in the reagent bottle turned to black color, while the lower 2/3 part granules did not significantly change color (brown-yellow) after the 8-wk storage. The black and brown-yellow color PA-degrading granules were manually separated and re-inoculated into two identical sequencing batch reactors for reviving the PA degradation capability. After a 7d operation, both black and yellow granules restored their activities to the levels before storage, in terms of total organic carbon removal efficiency (100%), specific oxygen uptake rate (59 mg g VSS(-1) h(-1)), and adenosine triphosphate content (0.016 mg g VSS(-1)). This study demonstrated that aerobic granules grown on a complex substrate could tolerate storage conditions and rapidly restored their bioactivities toward the target pollutant. The results also shed the light on the future application of precultured aerobic granules with unique functions for biodegradation and bioremediation purpose.

Granulicoccus phenolivorans gen. nov., sp. nov., a Gram-positive, phenol-degrading coccus isolated from phenol-degrading aerobic granules.

A Gram-positive bacterium, designated strain PG-02(T), was isolated by serial dilution from aerobic granules obtained from a laboratory-scale sequencing batch reactor for bioremediation of phenolic wastewater. Strain PG-02(T) grew axenically as cocci and is an oxidase-negative and catalase-positive, non-motile facultative anaerobe. It does not reduce nitrate and grows between 15 and 37 degrees C, with an optimum temperature of 30 degrees C. The pH range for growth is between 5.0 and 8.5, with an optimum pH of 7.0. Strain PG-02(T) contains type A3gamma peptidoglycan (ll-A(2)pm<--Gly with alanine at position 1 of the peptide subunit). The G+C content of the DNA is 69 mol%. Menaquinone MK-9(H(4)) was the major isoprenoid quinone. The polar lipids included diphosphatidylglycerol and phosphatidylglycerol, while 13-methyltetradecanoic acid (i-C(15 : 0)) and 1,1-dimethoxy-iso-pentadecane (i-C(15 : 0) DMA) were the major components in whole-cell methanolysates. PG-02(T) stained positively for intracellular polyphosphate granules but not poly-beta-hydroxyalkanoates. It produces capsular material and possesses an autoaggregation capability. Phenotypic and 16S rRNA gene sequence analyses showed that PG-02(T) differed from its closest phylogenetic relatives, namely members of the suborder Propionibacterineae, which includes the genera Tessaracoccus, Microlunatus, Luteococcus, Micropruina, Propionibacterium, Propioniferax, Nocardioides, Friedmanniella and Aeromicrobium, and that it should be placed in a new genus and species as Granulicoccus phenolivorans gen. nov., sp. nov. The type strain of Granulicoccus phenolivorans is PG-02(T) (=ATCC BAA-1292(T)=DSM 17626(T)).

Enhanced phenol biodegradation and aerobic granulation by two coaggregating bacterial strains.

The effect of coaggregation of the two bacterial strains Propioniferax-like PG-02 and Comamonassp. PG-08 on phenol degradation and aerobic granulation was investigated. While PG-02 was characterized as a phenol-degrader with a low half-saturation kinetics constant, PG-08 possessed strong aggregation ability with poor phenol degradation ability. The two strains coaggregated through involvement of lectin-saccharide interactions with the adhesin protein on strain PG-02 and the complementary sugar receptor on strain PG-08. Using the V. harveyi reporter strain BB170, it was found that both strains could produce autoinducer-2-like signals. If incubated together, the two strains showed cooperation for phenol degradation. In batch, the coculture degraded phenol at an initial concentration of 250 mg L(-1), faster than each strain separately. Bioaugmentation with simultaneously the two strains in sequencing batch reactors significantly improved phenol removal and aerobic granulation as compared to monoculture bioaugmentation. Bacterial coaggregation might be an integral component of the aerobic granulation process. Investigation of in situ occurrence of coggregation in aerobic granulation would help unveil its molecular mechanism. Then the granulation process could be improved through selection of specific microbial groups.

Physiological traits of bacterial strains isolated from phenol-degrading aerobic granules.

The physiological characteristics of ten bacterial strains isolated from phenol-degrading aerobic granules were evaluated in order to identify competitive traits for dominant growth in aerobic granules. The ten strains showed a wide diversity in specific growth rates and oxygen utilization kinetics, and could be divided into four catabolic types of phenol degradation. While some strains degraded phenol mainly via the meta pathway or the ortho pathway, other strains degraded phenol via both these pathways. The ten strains also exhibited high levels of autoaggregation and coaggregation activity. Within the collection of ten strains, 36.7% of all possible strain pairings displayed a measurable degree of coaggregation. Strain PG-08 possessed the strongest autoaggregation activity and showed significant coaggregation (coaggregation indices of 67% to 74%) with PG-02. The three strains PG-01, PG-02 and PG-08 belonging to dominant groups in the granules possessed different competitive characteristics. Microcosm experiments showed the three strains could not coexist at the high phenol concentration of 250 mg L(-1), but could coexist at lower phenol concentrations in a spatially heterogeneous environment. This study illustrated that the spatial heterogeneity provided by the aerobic granules led to niche differentiation and increased physiological diversity in the resident microbial community.

Biodegradation of p-nitrophenol by aerobic granules in a sequencing batch reactor.

In this study, aerobic granules to treat wastewater containing p-nitrophenol (PNP) were successfully developed in a sequencing batch reactor (SBR) using activated sludge as inoculum. A key step was the conditioning of the activated sludge seed to enrich for biomass with improved settleability and higher PNP degradation activity by implementing progressive decreases in settling time and stepwise increases in PNP concentration. The aerobic granules were cultivated at a PNP loading rate of 0.6 kg/ m3 x day, with glucose to boost the growth of PNP-degrading biomass. The granules had a clearly defined shape and appearance, settled significantly faster than activated sludge, and were capable of nearly complete PNP removal. The granules had specific PNP degradation rates that increased with PNP concentration from 0 to 40.1 mg of PNP/L, peaked at 19.3 mg of PNP/(g of VSS) x h (VSS = volatile suspended solids), and declined with further increases in PNP concentration as substrate inhibition effects became significant. Batch incubation experiments show that the PNP-degrading granules could also degrade other phenolic compounds, such as hydroquinone, p-nitrocatechol, phenol, 2,4-dichlorophenol, and 2,6-dichlorophenol. The PNP-degrading granules contained diverse microbial morphotypes, and PNP-degrading bacteria accounted for 49% of the total culturable heterotrophic bacteria. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments showed a gradual temporal shift in microbial community succession as the granules developed from the activated sludge seed. Specific oxygen utilization rates at 100 mg/L PNP were found to increase with the evolution of smaller granules to large granules, suggesting that the granulation process can enhance metabolic efficiency toward biodegradation of PNP. The results in this study demonstrate that it is possible to use aerobic granules for PNP biodegradation and broadens the benefits of using the SBR to target treatment of toxic and recalcitrant organic compounds.

Bioaugmentation and enhanced formation of microbial granules used in aerobic wastewater treatment.

Microbial aggregates of an aerobic granular sludge can be used for the treatment of industrial or municipal wastewater, but their formation from a microbial activated sludge requires several weeks. Therefore, the aim of this research was the selection of microbial cultures to shorten the granule-forming period from several weeks to a few days. An enrichment culture with the ability to accelerate granulation was obtained by repeating the selection and batch cultivation of fast-settling microbial aggregates isolated from the aerobic granular sludge. Bacterial cultures of Klebsiella pneumoniae strain B and Pseudomonas veronii strain F, with self-aggregation indexes of 65 and 51%, respectively, and a coaggregation index of 58%, were isolated from the enrichment culture. A mixture of these strains with the activated sludge was used as an inoculum in an experimental sequencing batch reactor to start up an aerobic granulation process. Aerobic granules with a mean diameter of 446+/-76 microm were formed in an experiment after 8 days of cultivation, but microbial granules were absent in controls. Considering biosafety issues, K. pneumoniae strain B was excluded from further studies, but P. veronii strain F was selected for larger-scale testing.

Quadrisphaera granulorum gen. nov., sp. nov., a Gram-positive polyphosphate-accumulating coccus in tetrads or aggregates isolated from aerobic granules.

A Gram-positive bacterium, designated strain AG019(T), was isolated by micromanipulation from aerobic granules obtained from a laboratory-scale sequencing batch reactor. This isolate grew axenically as cocci clustered predominantly in tetrads, and was morphologically similar to the dominant organisms observed in the biomass. The morphology also resembled that of the tetrad-forming organisms commonly seen in activated sludge samples. Strain AG019(T) was found to be an oxidase-negative, catalase-positive, non-motile aerobe that does not reduce nitrate and grows at temperatures between 15 and 40 degrees C, with an optimum at 37 degrees C. The pH range for growth was 5.0-9.0, with an optimum at pH 7.5. Strain AG019(T) contained a peptidoglycan with directly cross-linked meso-diaminopimelic acid (type A1gamma) and lacked mycolic acids. The G+C content of the DNA was 75 mol%. Menaquinone MK-8(H(2)) was the major isoprenoid quinone. The bacterium stained positively for intracellular polyphosphate granules but not for poly-beta-hydroxyalkanoates. It produced capsular material and showed autoaggregation ability. Phenotypic and 16S rRNA gene analyses showed that the bacterium differed sufficiently from its closest phylogenetic relatives, namely members of the suborder Frankineae, which includes the genera Geodermatophilus, Blastococcus, Frankia, Sporichthya, Acidothermus and Microsphaera, that it is proposed that it be placed in a novel genus, Quadrisphaera, as Quadrisphaera granulorum gen. nov., sp. nov. The type strain is AG019(T) (=ATCC BAA-1104(T)=DSM 44889(T)).

Start-up, microbial community analysis and formation of aerobic granules in a tert-butyl alcohol degrading sequencing batch reactor.

We demonstrate that compact well-settling aerobic granules can be developed in a sequencing batch reactor (SBR) for the biological removal of tert-butyl alcohol (TBA) using a strategy involving step increases in TBA loading rate achieved through increasing TBA concentrations in the influent. A moderate selection pressure that included a cycle time of 24 h and a start-of-cycle TBA concentration of 100 mg/L was initially introduced to encourage the growth and retention of biomass and avoid biomass loss from hydraulic washout. Start-of-cycle TBA concentrations were increased to 150, 300, 450, and 600 mg/L on days 90, 100, 121, and 199, respectively. These increases were only introduced after complete TBA removal was accompanied by visible improvements in biomass concentration and biomass settling ability. This acclimation strategy produced incrementally higher biomass concentrations and better settling biomass with higher specific TBA biodegradation rates. Effluent TBA concentrations were consistently below the detection limit of 25 microg/L. Aerobic granules were first observed about 180 days after reactor start-up. The granules had a clearly defined shape and appearance, settled significantly faster than the suspended sludge in the reactor, and eventually became the dominant form of biomass in the reactor. The adapted granules were capable of complete TBA removal and contained a stable microbial population with a low diversity of sequences of community 16S rRNA gene fragments. This study indicates that it is possible to use aerobic granules for TBA remediation and will contribute to a better understanding of how microbial acclimation can be exploited in the SBR to biologically remove recalcitrant xenobiotics.

Microbial adaptation to biodegradation of tert-butyl alcohol in a sequencing batch reactor.

This study demonstrates the utility of the sequencing batch reactor (SBR) to adapt microorganisms towards biological removal of tert-butyl alcohol (TBA). The reactor was inoculated with activated sludge and fed with TBA as the sole carbon source. Start-of-cycle TBA concentrations were initially set at 100 mgL(-1) with a cycle time of 24 h and a volumetric exchange ratio of 50% to maintain a TBA loading rate of not more than 100 mgL(-1)d(-1). Step increases in TBA loading rates up to 600 mgL(-1)d(-1) were achieved by first raising the start-of-cycle TBA concentration to 150 mgL(-1) on day 90 and subsequently by reducing the cycle time from 24 to 12, 8 and 6h on days 100, 121 and 199, respectively. This acclimation strategy favored the retention of increasingly higher densities of well-adapted microbial populations in the reactor. The increases in TBA loading produced better settling biomass and higher biomass concentrations with higher specific TBA biodegradation rates. Effluent TBA concentrations were consistently below the detection limit of 25 microgL(-1). The use of progressively shorter cycle times created selection pressures that fostered the self-immobilization of the reactor microorganisms into aerobic granules which first appeared on day 125. Specific TBA biodegradation rates in the granules followed the Haldane model for substrate inhibition, and peaked at 13.8 mgTBAgVSS(-1)h(-1) at a TBA concentration of 300 mgL(-1). Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes from granules sampled between days 220 and 247 confirmed the existence of a highly stable microbial community with members belonging to the alpha, beta and delta subdivisions of Proteobacteria and the Cytophaga-Flavobacteria-Bacteroides (CFB) group.

Rapid cultivation of stable aerobic phenol-degrading granules using acetate-fed granules as microbial seed.

The aim of this study is to evaluate the utility of using aerobic acetate-fed microbial granules as a starting seed to rapidly develop stable aerobic phenol-degrading granules. Aerobic granules were first cultivated in four sequencing batch reactors with acetate as sole carbon source at a loading rate of 3.8 kg m(-3) d(-1). Phenol was then added to reactors R1, R2, R3 and R4 at loading rates of 0, 0.6, 1.2 and 2.4 kg m(-3) d(-1), respectively. The granules acclimated quickly to the phenol loading, and stabilized only 1 week after phenol was introduced. The granules exhibited good settling ability with good biomass retention and good metabolic activity, as evidenced by the low SVI values, stable biomass concentrations and good removal of acetate and phenol. No significant inhibitory effects from phenol toxicity were observed at the intermediate loadings of 0.6 and 1.2 kg phenol m(-3) d(-1), except for a slight lag in the ability of the granules to degrade phenol during the initial cycles. At the highest loading of 2.4 kg phenol m(-3) d(-1), a sharp buildup of phenol was observed in reactor R4 because the granules were initially unable to degrade phenol. However, this buildup quickly dissipated as the granules adapted rapidly to the high phenol concentrations. The compact structure of the acetate-fed granules likely protected the microorganisms against phenol toxicity and facilitated microbial acclimation towards faster phenol degradation rates. This is the first study to demonstrate the benefits of using aerobic granules cultivated on benign substrates as microbial seed to produce granules to degrade toxic substrates. This concept of using granules to produce different granules can be extended to granule-based applications involving other toxic chemicals and other types of high-strength industrial wastewaters, where rapid reactor start-up and system stability are key considerations.

Comparing activated sludge and aerobic granules as microbial inocula for phenol biodegradation.

Activated sludge and acetate-fed granules were used as microbial inocula to start up two sequencing batch reactors (R1, R2) for phenol biodegradation. The reactors were operated in 4-h cycles at a phenol loading of 1.8 kg m(-3) day(-1). The biomass in R1 failed to remove phenol and completely washed out after 4 days. R2 experienced initial difficulty in removing phenol, but the biomass acclimated quickly and effluent phenol concentrations declined to 0.3 mg l(-1) from day 3. The acetate-fed granules were covered with bacterial rods, but filamentous bacteria with sheaths, presumably to shield against toxicity, quickly emerged as the dominant morphotype upon phenol exposure. Bacterial adaptation to phenol also took the form of modifications in enzyme activity and increased production of extracellular polymers. 16S rRNA gene fingerprints revealed a slight decrease in bacterial diversity from day 0 to day 3 in R1, prior to process failure. In R2, a clear shift in community structure was observed as the seed evolved into phenol-degrading granules without losing species-richness. The results highlight the effectiveness of granules over activated sludge as seed for reactors treating toxic wastewaters.

Bacterial diversity and function of aerobic granules engineered in a sequencing batch reactor for phenol degradation.

Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of beta-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.

Thermoactive extracellular proteases of Geobacillus caldoproteolyticus, sp. nov., from sewage sludge.

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65 degrees C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH4Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0-11.0 and a temperature range of 40-90 degrees C, with an optimal pH of 8.0-9.0 and an optimal temperature of 70-80 degrees C, respectively. The protease was stable over the temperature range of 40-70 degrees C and retained 57 and 38% of its activity at 80 and 90 degrees C, respectively, after 1 h.

Intensive aerobic bioconversion of sewage sludge and food waste into fertiliser.

The aim of this research was to verify the possibility of recovering the nutrients present in sewage sludge and vegetable food waste as fertiliser after aerobic thermophilic intensive bioconversion. The process was performed in a closed reactor under controlled conditions of aeration, stirring and pH, at a temperature of 60 degrees C, after addition of a starter bacterial culture of Bacillus thermoamylovorans SW25. End product with the best fertilising properties was obtained when sewage sludge, mixed with food waste, CaCO3 and an artificial bulking agent was thermally pretreated. The content of volatile solids and organic carbon decreased from 82.8% to 62.3% and from 37.7% to 32.5% of total solids (TS) respectively, during 12 days of bioconversion. The stable organic fertiliser produced was a powder with moisture content of 5%. Furthermore, 3.4% of nitrogen, 0.4% of phosphorus and 2.9% of potassium were also present. Addition of 10-15g of this fertiliser to 1 kg of poor fertility soil increased the growth of different plants by 113-164%.

Ca2+ augmentation for enhancement of aerobically grown microbial granules in sludge blanket reactors.

Two sequential aerobic sludge blanket reactors were concurrently operated to examine the effect of Ca2+ augmentation on aerobic granulation. Augmentation with 100 mg Ca2+ l(-1) significantly decreased the time to cultivate aerobically grown microbial granules from 32 d to 16 d. Ca2+-fed granules were denser and more compact, showed better settling and strength characteristics, and had higher polysaccharide contents.