Understanding the properties of aerobic sludge granules as hydrogels

Aerobic sludge granules are larger, denser microbial aggregates than activated sludge flocs with a smoother and more regular surface, which facilitates greater wastewater treatment intensity. Factors important in their growth are still poorly understood, which is an impediment to the construction and operation of full-scale aerobic sludge granule processes. Data in this article obtained with granules treating an abattoir wastewater provide evidence that aerobic sludge granules are hydrogels. The results also demonstrate a method for characterizing macromolecular associations. The rheological profile of these granules was found to be analogous with that of typical polymer gels. Water uptake or swelling reflects an equilibrium between granule elastic modulus and osmotic pressure, whereby uptake is increased by reducing solute concentration or the elastic modulus. A weakening of the extracellular polymeric substance (EPS) matrix as demonstrated with mechanical spectroscopy was induced by several environmental factors including temperature, pH and ionic strength. Uniform and elastic deformation was observed at low strain. Enzymatic degradation studies indicate that proteins and alpha-polysaccharides were the major granule structural materials. The aerobic sludge granules in the current study were therefore protein-polysaccharide composite physical hydrogels. While aerobic sludge granules treating an abattoir wastewater are used as a case study, many of the fundamental principles detailed here are relevant to other granulation processes. The paradigm established in this study can potentially be applied to better understand the formation of aerobic sludge granules and thus overcome a hurdle in the acceptance of aerobic sludge granulation as an alternative to more traditional wastewater treatment processes.     

Specific aerobic granules can be developed in a completely mixed tank reactor by bioaugmentation using micro-mycelial pellets of Phanerochaete chrysosporium

Aerobic granules were firstly developed in a completely mixed tank reactor (CMTR) by seeding micro-mycelial pellets (MMPs) of Phanerochaete chrysosporium. During phenol wastewater treatment, sludge granulation rate reached 67 % after 15-day operation. The granules in CMTR are different from aerobic granules described in literature in morphology, and a majority of them are rod-shaped or rodlike sludge besides spherical granules. The polymorphic granules, having no essential difference with aerobic granules previously reported, achieve advantages over conventional activated sludge in settling ability, biomass concentration, density, integrity coefficient and removal ability to phenol wastewater. The optimized parameters for sludge granulation in CMTR including temperature, inoculum quantity, rotary speed and superficial air upflow velocity are 30 °C, 5–7 g/l, 150 rpm, and 0.5 cm/s, respectively. Analysis on sludge granulation mechanism indicates that MMPs not only result in the formation of aerobic granules containing MMPs as nuclei, but also induce the formation of biogranules which do not have MMP at their cores. The work challenges the general belief that the homogenous circular flow pattern of microbial aggregates is necessary for aerobic sludge granulation.     

Biomass and porosity profiles in microbial granules used for aerobic wastewater treatment

AIMS: To obtain biomass and porosity profiles for aerobically grown granules of different diameters and to determine a suitable range of granule diameters for application in wastewater treatment. METHODS AND RESULTS: Microbial granules were cultivated in an aerobic granulated sludge reactor with model wastewaters containing acetate, or ethanol plus acetate, or glucose as the main carbon source. Granules were formed by retaining microbial aggregates using a settling time of 2 min. Sampled granules had diameters ranging from 0.45 to 3 mm. Microbial biomass in the granules was detected with the nucleic acid stain SYTO 9 and confocal laser scanning microscopy. The thickness of the microbial biomass layer was proportional to the granule diameter, and had a maximum value of 0.8 mm. The thickness of the microbial biomass layer correlated with the penetration depth of 0.1 microm fluorescent beads into the granule. CONCLUSIONS: The microbial biomass and porosity studies suggest that aerobically grown microbial granules should have diameters less than a critical diameter of 0.5 mm, if deployed for wastewater treatment applications. This critical diameter is based on the assumption that whole granules should have a porous biomass-filled matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could contribute to the development of aerobic granulation technology for effective biological wastewater treatment.     

Physicochemical characteristics of microbial granules

Microbial granules play an important role in the field of biological wastewater treatment due to their advantages over the conventional sludge flocs, such as a denser and stronger aggregate structure, better settleability and ensured solid-effluent separation, higher biomass concentration, and greater ability to withstand shock loadings. A better understanding of microbial granules may help in engineering biological wastewater treatment systems. Recent studies have greatly expanded our vision over the physicochemical characteristics of microbial granules. This paper provides an up-to-date review on recent work in the understanding of physicochemical characteristics of both anaerobic and aerobic granules with regard to settleability, permeability, morphology, mechanical stability, rheology, porosity, surface adsorbability, surface hydrophobicity and thermodynamics, and extracellular polymeric substances. Our growing knowledge on such characteristics might facilitate the engineering and optimization of microbial granulation as one of the most promising techniques in biological wastewater treatment.     

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.     

Aerobic granules: a novel zinc biosorbent

AIMS: Aerobic granules are aggregates with a compact and porous microbial structure. In view of the potential use of aerobic granules as biosorbents for Zn(II) removal from industrial wastewater, this study investigated the effects of initial Zn(II) and aerobic granule concentrations on the kinetics of Zn(II) biosorption on the aerobic granule surface. METHODS AND RESULTS: Acetate-fed aerobic granules with a mean diameter of 1.0 mm were used as biosorbents. Results showed that the kinetics of Zn(II) biosorption on the aerobic granule surface were related to both initial Zn(II) and granule concentrations. It was found that the real driving force for Zn(II) biosorption on the aerobic granule surface could be described by the ratio of initial Zn(II) concentration (Co) to initial granule concentration (Xo), rather than individual Co or Xo. The Co/Xo ratio provides a unified basis for interpretation of the biosorption data obtained under different initial conditions. The maximum biosorption capacity of Zn(II) by aerobic granules was 270 mg g(-1). CONCLUSIONS: It appears that the aerobic granule can be used as an effective biosorbent for efficient removal of Zn(II) or other types of heavy metals from industrial wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: This study could lead to the development of a novel granular biosorbent for the removal of heavy metals from wastewater. A simple and compact aerobic granule-based biosorber could be expected.     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

Compound versus multigranular exocytosis in peritoneal mast cells

We have used the whole-cell patch-pipette technique to measure the step increases in the cell membrane capacitance (equivalent to the membrane area) caused by the fusion of secretory granules in degranulating murine mast cells. We have observed that up to 30% of the total membrane expansion caused by degranulation results from large fusion events that cannot be explained by the fusion of single secretory granules. These large events are observed mainly in the initial phase of a degranulation. We have developed a simple mathematical model for a mast cell to test whether these large events are caused by a stimulus-induced, granule-to-granule fusion that occurs before their exocytosis (multigranular exocytosis). Our results suggest that the large fusion events are caused by the exocytosis of granule aggregates that existed before stimulation and that are located at the cell's periphery. We propose a novel mechanism by which granule aggregates can be formed at the periphery of the cell. This mechanism relies on the ability of a transiently fused granule ("flicker") to fuse with more internally located granules in a sequential manner. This pattern may result in the formation of larger peripheral granules that later on can fuse with the membrane. The formation of peripheral granule aggregates may potentiate a subsequent secretory response.     

Osmoregulated periplasmic glucans of Erwinia chrysanthemi

We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of Erwinia chrysanthemi. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000-0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and (1)H and (13)C nuclear magnetic resonance analyses. OPGs produced by E. chrysanthemi are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of beta-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by beta-1,6 linkages in a random way. This glucan backbone may be substituted by O-acetyl and O-succinyl ester-linked residues.     

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.     

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 β-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.     

Microscopic observation of aerobic granulation in sequential aerobic sludge blanket reactor

AIMS: This paper attempts to provide visual evidence of how aerobic granulation evolves in sequential aerobic sludge blanket reactors. METHODS AND RESULTS: A series of experiments were conducted in two column-type sequential aerobic sludge reactors fed with glucose and acetate as sole carbon source, respectively. The evolution of aerobic granulation was monitored using image analysis and optical and scanning electron microscopy. The results indicated that the formation of aerobic granules was a gradual process from seed sludge to compact aggregates, further to granular sludge and finally to mature granules with the sequential operation proceeding. Glucose- and acetate-fed granules have comparable characteristics in terms of settling velocity, size, shape, biomass density and microbial activity. However, the microbial diversity of the granules was associated with the carbon source supplied. In this work, an important aerobic starvation phase was identified during sequential operation cycles. It was found that periodical aerobic starvation was an effective trigger for microbial aggregation in the reactor and further strengthened cell-cell interaction to form dense aggregates, which was an essential step of granulation. The periodical starvation-induced aggregates would finally be shaped to granules by hydrodynamic shear and flow. CONCLUSION: Aerobic granules can be formed within 3 weeks in the systems. The periodical starvation and hydrodynamic conditions would play a crucial role in the granulation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic granules have excellent physical characteristics as compared with conventional activated sludge flocs. This research could be helpful for the development of an aerobic granule-based novel type of reactor for handling high strength organic wastewater.     

Carbon,nitrogen and phosphorus removal mechanisms of aerobic granules

Aerobic granules are the potential tools to develop modern wastewater treatment technologies with improved nutrient removal efficiency. These granules have several promising advantages over conventional activated sludge-based wastewater treatment processes. This technology has the potential of reducing the infrastructure and operation costs of wastewater treatment by 25%, energy requirement by 30%, and space requirement by 75%. The nutrient removal mechanisms of aerobic granules are slightly different from that of the activated sludge. For instance, unlike activated sludge process, according to some reports, as high as 70% of the total phosphorus removed by aerobic granules were attributed to precipitation within the granules. Similarly, aerobic granule-based technology reduces the total amount of sludge produced during wastewater treatment. However, the reason behind this observation is unknown and it needs further explanations based on carbon and nitrogen removal mechanisms. Thus, as a part of the present review, a set of new hypotheses have been proposed to explain the peculiar nutrient removal mechanisms of the aerobic granules.     

Nuclear magnetic resonance studies of blood platelets

Blood platelets contain membrane-enclosed granules which have inside them high concentrations of 5-hydroxytryptamine (serotonin) along with adenine nucleotides and divalent metal ions. 19F n.m.r. of fluorinated serotonin incorporated into the granules of both human and pig intact platelets has shown that the motional state of the serotonin is restricted. Comparison with 31P n.m.r. experiments indicates that this restriction of motion is a consequence of high molecular weight aggregates formed by the adenine nucleotides and metal ions, and that it varies with the species from which the platelets are obtained. In the case of human platelet granules, at least, these high molecular weight aggregates are present in the absence as well as in the presence of serotonin. The biological significance of these data is briefly discussed.     

Structural analysis of Escherichia coli OpgG, a protein required for the biosynthesis of osmoregulated periplasmic glucans

Osmoregulated periplasmic glucans (OPGs) G protein (OpgG) is required for OPGs biosynthesis. OPGs from Escherichia coli are branched glucans, with a backbone of beta-1,2 glucose units and with branches attached by beta-1,6 linkages. In Proteobacteria, OPGs are involved in osmoprotection, biofilm formation, virulence and resistance to antibiotics. Despite their important biological implications, enzymes synthesizing OPGs are poorly characterized. Here, we report the 2.5 A crystal structure of OpgG from E.coli. The structure was solved using a selenemethionine derivative of OpgG and the multiple anomalous diffraction method (MAD). The protein is composed of two beta-sandwich domains connected by one turn of 3(10) helix. The N-terminal domain (residues 22-388) displays a 25-stranded beta-sandwich fold found in several carbohydrate-related proteins. It exhibits a large cleft comprising many aromatic and acidic residues. This putative binding site shares some similarities with enzymes such as galactose mutarotase and glucodextranase, suggesting a potential catalytic role for this domain in OPG synthesis. On the other hand, the C-terminal domain (residues 401-512) has a seven-stranded immunoglobulin-like beta-sandwich fold, found in many proteins where it is mainly implicated in interactions with other molecules. The structural data suggest that OpgG is an OPG branching enzyme in which the catalytic activity is located in the large N-terminal domain and controlled via the smaller C-terminal domain.     

Degradation of methanethiol by methylotrophic methanogenic archaea in a lab-scale upflow anaerobic sludge blanket reactor

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30 degrees C to H2S, CO2, and CH4. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter-1 day-1. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.     

Role of anionic charges of osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium SL1344 in mice virulence

opgB gene of Salmonella enterica serovar Typhimurium was identified earlier in a genome-wide screen for mice virulence (Valentine et al. in Infect Immun 66:3378-3383, 1998). Although mutation in opgB resulted in avirulent Salmonella strain, how this gene contributes to pathogenesis remains unclear. Based on DNA homology, opgB is predicted to be responsible for adding phosphoglycerate residues to osmoregulated periplasmic glucans (OPGs) giving them anionic characteristics. In Escherichia coli, yet another gene, opgC, is also reported to contribute to anionic characteristics of OPGs by adding succinic acid residues. We constructed opgB, opgC, and opgBC double mutants of S. enterica serovar Typhimurium strain SL1344. As predicted opgBC mutant synthesized neutral OPGs that were devoid of any anionic substituents. However, opgB, opgC, and opgBC mutations had no significant impact on mice virulence as well as on competitive organ colonization. In low osmotic conditions, opgB, opgC, and opgBC mutants exhibited delay in growth initiation in the presence of sodium deoxycholate. Anionic substituents of OPGs from Salmonella although appear to be needed to overcome resistance of deoxycholate in hypoosmotic growth media, no evidence was found for their role in mice virulence.     

Glycosomes (protein-glycogen complex) in the canine heart. Ultrastructure, histochemistry and changes induced by acidic treatment.

Cardiac conducting fibers were selected from two dogs defined as A and B. The specimens differed in the reaction of their electron dense granules, commonly referred to as glycogen, to the treatment en bloc with uranyl acetate. Material was fixed in glutaraldehyde and OsO4. Blocks were processed either conventionally or immersed in uranyl acetate before dehydration. Sections were examined unsatined, stained with U and/or Pb or with a histochemical technique (PA-TSC-SP) specific for glycogen. Electron dense granules have affinity to Os, U and Pb which suggests ionic reactions specific for protein but improbable for glycogen. Large granules in A turned into pale ghosts and small granules in B disappeared after treatment en bloc with uranyl acetate. PA-TSC-SP in conventional samples showed glycogen particles arranged into aggregates corresponding in size to the electron dense granules. Treatment en bloc slightly affected glycogen aggregates in A and resulted in a formation of large clumps of glycogen particles in B. It was concluded that the electron dense granules represented protein bound to glycogen in the organelles called glycosomes. Acidic action of uranyl acetate removed protein from glycosomes. The degree of this removal depended on the amount of protein present in glycosomes in the moment of fixation.     

Osmoregulated periplasmic glucans in Proteobacteria

Large amounts of osmoregulated periplasmic glucans (OPGs) are found in the periplasmic space of Proteobacteria. Four families of OPGs are described on the basis of structural features of the polyglucose backbone. Depending on the species considered, OPGs can be modified to various extent by a variety of substituents. Genes governing the backbone synthesis are identified in a limited number of species. They belong to three unrelated families. OPG synthesis is subject to osmoregulation and feedback control. Osmoregulation can occur at the level of gene expression and/or at the level of enzyme activity. Mutants defective in OPG synthesis have a highly pleiotropic phenotype, indicative of an overall alteration of their envelope properties. Mutants of this kind were obtained as attenuated or avirulent derivatives of plant or animals pathogen. Thus, OPGs appear to be important intrinsic components of the Gram-negative bacterial envelope, which can be essential in extreme conditions found in nature, and especially when bacteria must interact with an eukaryotic host.