The effects of extracellular polymeric substances on the formation and stability of biogranules

Biogranulation is a promising biotechnology developed for wastewater treatment. Biogranules exhibit a matrix microbial structure, and intensive research has shown that extracellular polymeric substances (EPS) are a major component of the biogranule matrix material in both anaerobic and aerobic granules. This paper aims to review the role of EPS in biogranulation, factors influencing EPS production, the effect of EPS on cell surface properties of biogranules, and the relationship of EPS to the structural stability of biogranules. EPS production is substantially enhanced when the microbial community is subject to stressful culture conditions, and the stimulated EPS production in the microbial matrix in turn favours the formation of anaerobic and aerobic granules. EPS can also play an essential role in maintaining the integrity and stability of spatial structure in mature biogranules. It is expected that this paper can provide deep insights into the functions of EPS in the biogranulation process.     

Distribution of EPS and cell surface hydrophobicity in aerobic granules

This study described the distribution of extracellular polysaccharides (EPS) and hydrophobicity in aerobic granule as well as the essential role of EPS in maintaining the stable structure of aerobic granules. Aerobic granules showed a heterogeneous structure, which had an outer shell with high biomass density and an inner core having a relatively low biomass density. Results showed that the outer shell of aerobic granule was composed of poorly soluble and noneasily biodegradable EPS, whereas its core part was filled with readily soluble and biodegradable EPS. It was further found that the shell of aerobic granule exhibited a higher hydrophobicity than the core of granule. The insoluble EPS present in the granule shell would play a protective role with respect to the structure stability and integrity of aerobic granules.     

Long-term storage and subsequent reactivation of aerobic granules

This study investigated a seven month storage and the subsequent reactivation of aerobic granules. The granule size and structure integrity were remained during storage, whereas some cavities and pleats appeared on the surface and further deteriorated the settleability. Along with the reactivation, the physical characteristics and microbial activities of aerobic granules were gradually improved. Activities of heterotrophs and nitrifiers can be fully recovered within 16days and 11days, respectively. Nitrifiers decayed slower during storage and reinstated rapider during reactivation than heterotrophs. In fresh aerobic granules, the dominated ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were Nitrosomonas and Nitrospira, respectively. During storage, the initially dominated populations decayed rapider than the initially less dominated ones. Extracellular polymeric substances (EPS) significantly decreased within the first month, and then gradually accumulated during the last six months storage. Accumulation of EPS was an effective strategy for maintaining structural integrity of aerobic granules during long-term storage.     

Biodegradability of extracellular polymeric substances produced by aerobic granules

This study investigated the biodegradability of extracellular polymeric substances (EPS) produced by aerobic granules. Aerobic granules were precultivated with synthetic wastewater in a lab-scale sequencing batch reactor. EPS were extracted from aerobic granules and were then fed as the sole carbon source to their own producers. Results showed that about 50% of EPS produced by aerobic granules could be utilized by their producers under aerobic starvation condition. The average biodegradation rate of the granule EPS in terms of chemical oxygen demand was five times slower than that of acetate, but 50 times faster than that of nonbiodegradable EPS produced by aerobic granules. The nonbiodegradable EPS was mainly found on the outer shell of aerobic granule. EPS produced by aerobic granules basically comprised two major components, i.e., biodegradable and nonbiodegradable EPS. The biodegradable EPS could serve as a useful energy source to sustain the growth of aerobic granules under starvation. This study provides experimental evidence that part of the EPS produced by aerobic granules would be biodegradable, but only nonbiodegradable EPS would play a crucial role in maintaining the structural integrity of aerobic granule.     

Role of N-acyl homoserine lactone (AHL)-based quorum sensing (QS) in aerobic sludge granulation

N-acyl homoserine lactone (AHL)-based quorum sensing (QS) has been recognized to play an important role in the formation of biofilm. However, aerobic granular sludge is considered as a special biofilm, and its biological implication and role of AHL-based QS still remain unclear. This study investigated the role of AHL-based QS in aerobic granulation. Results showed that AHLs were necessary to the typical aerobic granulation, and AHL-associated coordination of bacteria in sludge aggregation was sludge density dependent only when it reached a threshold of 1.010 g/mL; AHL-based QS was activated to regulate aerobic granulation. Furthermore, a quorum quenching method was firstly adopted to investigate the role of AHLs in aerobic granules. Results showed inhibition of AHL by acylase that reduced the AHL content in aerobic granules and further weakened its attachment potential, which proved that AHLs play an important role in the formation of aerobic granules. Additionally, the assay of quorum quenching not only proved that AHL-based QS could regulate EPS production but also provided additional evidence for the role of AHLs in aerobic granulation by regulating EPS content and its component proportion.     

Composition and Distribution of Extracellular Polymeric Substances in Aerobic Flocs and Granular Sludge

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-μm cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 ± 12 ml g⁻¹, and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 ± 2 ml g⁻¹. EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.     

Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-mum cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 +/- 12 ml g(-1), and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 +/- 2 ml g(-1). EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.     

The influence of short-term starvation on aerobic granules

Evidence shows that almost all aerobic granules can only be cultivated in sequencing batch reactor (SBR). Compared to continuous process, the unique feature of SBR is its cycle operation, which results in a periodical starvation in the reactor. So far, the effect of such a periodical starvation on aerobic granulation process remains unknown. Thus, this study investigated the responses of aerobic granules to the respective carbon-, nitrogen-, phosphorus-, potassium-starvation and also their collective effects in terms of cell surface hydrophobicity, surface zeta potential, extracelluar polysaccharides content, specific oxygen utilization rate and biomass growth. Results showed that short-term C-, N-, P- and K- starvations would pose negative effects on aerobic granules, e.g. reduce EPS content, inhibit microbial activity, weaken structural integrity and worsen settleability of aerobic granules. This study likely provides primary evidence that the substrate and nutrients starvation would not contribute to the stability of aerobic granules in a significant way.     

Energy uncoupling inhibits aerobic granulation

Although aerobic granulation has been intensively studied, the possible mechanism of this cell-to-cell self-immobilization phenomenon still remains unclear. Aerobic granulation in the absence and presence of a chemical uncoupler, 3,3′,4′,5-tetrachlorosalicylanilide (TCS), which can dissipate the proton gradient and further disrupt ATP synthesis, was investigated. Upon exposure to TCS, precultivated mature aerobic granules underwent disintegration, indicating that the stability and integrity of aerobic granules would be associated with microbial energy metabolism. It was also shown that the formation of aerobic granules in the presence of TCS was completely inhibited as compared with the control free of TCS. These results, for the first time, reveal that aerobic granulation is energy metabolism dependent, and possible reasons are also discussed.     

Effects of long-term addition of Cu(II) and Ni(II) on the biochemical properties of aerobic granules in sequencing batch reactors

Copper (Cu(II)) and nickel (Ni(II)) are often encountered in wastewaters. This study investigated the individual toxic effects of long-term addition of Cu(II) and Ni(II) on the biochemical properties of aerobic granules in sequencing batch reactors (SBRs). The biochemical properties of aerobic granules were characterized by extracellular polymeric substances (EPS) content, dehydrogenase activity, microbial community biodiversity, and SBR performance. One SBR was used as a control system, while another two received respective concentration of Cu(II) and Ni(II) equal to 5 mg/L initially and increased to 15 mg/L on day 27. Results showed that the addition of Cu(II) drastically reduced the biomass concentration, bioactivity, and biodiversity of aerobic granules, and certainly deteriorated the treatment performance. The toxic effect of Ni(II) on the biodiversity of aerobic granules was milder and the aerobic granular system elevated the level of Ni(II) toxicity tolerance. Even at a concentration of 15 mg/L, Ni(II) still stimulated the biomass yield and bioactivity of aerobic granules to some extent. The elevated tolerance seemed to be owed to the concentration gradient developed within granules, increased biomass concentration, and promoted EPS production in aerobic granular systems.     

Distribution of extracellular polymeric substances in aerobic granules

Extracellular matrix provides an architectural structure and mechanical stability for aerobic granules. Distributions of cells and extracellular polymeric substances (EPS), including proteins, α- and β-d-glucopyranose polysaccharides, in acetate-fed granules and phenol-fed granules were probed using a novel quadruple staining scheme. In acetate-fed granules, protein and β-d-glucopyranose polysaccharides formed the core, whereas, the cells and α-d-glucopyranose polysaccharides accumulated in the granule outer layers. Based on these experimental findings, this study indicated that different conclusions can be obtained regarding EPS distributions when granules were stained differently. The core of phenol-fed granules, conversely, was formed principally by proteins; whereas, the cells and α- and β-d-glucopyranose polysaccharides were accumulated at an outer filamentous layer. Using a series of confocal laser scanning microscope (CLSM) images whose threshold values were determined via Otsu’s scheme, the three-dimensional distributions of cells and EPS were produced using a polygonal surface model. Structural information extracted can be applied in further development of comprehensive granule models.     

Formation of filamentous aerobic granules: role of pH and mechanism

Filamentous overgrowth in aerobic granular sludge processes can cause reactor failure. In this work, aerobic granules were cultivated in five identical sequencing batch reactors with acetate or glucose as the carbon source with various values of influent pH (4.5–8). Microscopic observations revealed that acidic pH, rather than the species of carbon source, epistatically controls the aerobic granules with filamentous structure. An acidic pH shifted the structure of the microbial community in the granules, such that the fungus Geotrichum fragrans was the predominant filamentous microorganism therein. The acidic pH reduced the intracellular cyclic diguanylate (c-di-GMP) content for increasing the motility of the bacteria to washout and increase the growth rate of G. fragrans on glucose or acetate, together causing overgrowth of the fungus. Maintaining the suspension under alkaline condition is proposed as an effective way to suppress filamentous overgrowth and maintain granule stability.     

Improved stability of aerobic granules by selecting slow-growing nitrifying bacteria

This study investigated the feasibility of improving the stability of aerobic granules through selecting slow-growing nitrifying bacteria. For this purpose, four sequencing batch reactors were operated at different substrate N/COD ratios ranging from 5/100 to 30/100. Results showed that aerobic granules formed in all four reactors, and aerobic granulation was a gradual process evolving from the dispersed seed sludge to mature and stable granules, and the whole granulation process could be divided into three phases, i.e. acclimation phase, granulation followed by granule maturation. The observed growth rate and mean size of mature aerobic granules were found to decrease as the substrate N/COD ratio was increased, while nitrifying population was enriched markedly in aerobic granules developed at high substrate N/COD ratios. The enriched nitrifying population in aerobic granules was responsible for the observed low growth rate of aerobic granules. It seems certain that the substrate N/COD ratio is an important factor in selecting nitrifying bacteria in aerobic granules. Aerobic granules with low growth rates showed strong structure and good settleability in terms of specific gravity, SVI and cell hydrophobicity that further lead to high stability as compared to those having high growth rates. This study demonstrated that the selection of slow-growing nitrifying bacteria through controlling substrate N/COD ratio would be a useful strategy for improving the stability of aerobic granules.     

High rate performance and characterization of granular methanogenic sludges in upflow anaerobic sludge blanket reactors fed with various defined substrates

High rate granular methanogenic fermentations were performed in one-phase upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewaters containing starch, sucrose, ethanol, and butyrate plus propionate. All granules formed showed high settling velocities which enabled high cell mass retention and accommodation of high loading rates. The maximum COD removal rates (g COD/l-reactor·d) obtained after 500-d operations were 7.6 for starch, 10.5 for sucrose, 32.1 for ethanol, and 42.6 for butyrate-propionate. Long-term growth on various defined substrates altered the population of bacterial trophic groups and overall characteristics of granules. The starch- and sucrose-grown granules were characterized by larger size and more abundant extracellular polymeric substances (EPS) than the ethanol- or fatty acids-grown granules. The fatty acids-grown granules contained a considerable amount of inorganic salts (ash content: 56 to 63%) but a small amount of EPS, and showed a denser ultrastructure than the other three types of granules. The granules grown on ethanol under slightly acidic conditions showed the lowest specific gravity and volatile suspended solids (VSS) density as well as ash content among all of the granules. As aceticlastic methanogens, Methanothrix spp. were predominant in the starch-, sucrose-, and fatty acids-grown granules, whereas comparable numbers of Methanosarcina spp. were observed only in the ethanol-grown granules. The populations of hydrogenotrophic methanogens were the largest of all bacterial trophic groups in the respective granules. The data confirm that the prevalence of Methanothrix spp. and high methanogenic activity for H₂ are general characteristics of methanogenic granucles and that EPS and inorganic deposits contribute chemically to the enhancement of structural stability and mechanical strength of granules.     

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.     

Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm

Aims: Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Methods and Results: Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR‐based denaturing gradient‐gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Conclusions: Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. Significance and Impact of the Study: The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.     

Stability of sludge flocs under shear conditions: roles of extracellular polymeric substances (EPS)

The roles of extracellular polymer substances (EPS) in the shear stability of aerobic and anaerobic flocs were investigated. Both pH and EDTA concentration had a significant effect on the floc stability. The sludge flocs became much weaker as the solution pH increase to above 10. Addition of 1 mM EDTA or more could cause considerable cell erosion and deflocculation of the anaerobic flocs, whereas more than 3 mM EDTA was needed to show its adverse effect on the stability of aerobic flocs. A fraction of the EPS, around 10 mg/g SS for the aerobic flocs and 15 mg/g SS for the anaerobic flocs, could be extracted by fluid shear when the dispersed mass concentration approached the equilibrium. This suggests that most of the dispersed particles were glued by a small amount of readily-extractable EPS fraction. In addition to the abundance of this EPS fraction, its proteins/carbohydrates ratio, about 0.22:1 for the aerobic flocs and 2.66:1 for the anaerobic flocs, also appeared to be an important factor governing the microbial floc stability. A lower content of the readily-extractable EPS fraction and a lower ratio of proteins/carbohydrates were responsible for the greater stability of microbial flocs. The total content of the EPS, however, did not show a direct correlation with the floc stability. A hypothesis about biological flocs with two distinct structural regions was proposed. The outer part contained dispersible cells loosely entangled by the readily-extractable EPS fraction. This part was layered and would become completely dispersed at an infinite shear intensity. On the other hand, the inner part contains biomass in a stable structure tightly glued by EPS, which could not be dispersed by shear except under unfavorable conditions.     

The quorum-sensing effect of aerobic granules on bacterial adhesion, biofilm formation, and sludge granulation

Quorum sensing (QS) through signal chemical molecules is known to be essential to bacterial adhesion and biofilm formation. In this study, the QS ability of aerobic granules—a special form of biofilms used for biological wastewater treatment—was investigated and compared with that of conventional activated sludge flocs. A novel sectional membrane bioreactor was used together with a flow-cell to evaluate the possible influence of signal chemicals produced by the source sludge on the growth mode of bacterial cells. The results demonstrate the apparent production of QS chemicals from granules and its impact on initial cell attachment and granule formation. When granules were used as the signal-producing biomass, the attached-growth mode was dominant for the free cells, and the biofilm formation rate in the flow-cell was about ten times faster than in cases which used activated sludge as the signal source biomass. In addition, the intracellular extract from mature granules significantly accelerated the sludge granulation process. It is argued that the production and expression of QS signal chemicals from granules and granule precursors might have induced the gene expression of bacteria in suspension for attached growth rather than suspended growth, leading to granule formation and its stable structure.     

Involvement of ATP and autoinducer‐2 in aerobic granulation

Aerobic granulation represents an important bacterium‐to‐bacterium self‐immobilization process that has been exploited for the treatment of a wide spectrum of wastewaters, but the mechanism behind still remains unclear in a microbiological sense. This study investigated the possible involvement of ATP and autoinducer‐2 (AI‐2) in aerobic granulation. Results revealed that initiation of microbial aggregation is closely associated with the ATP content of biomass, whereas AI‐2 of biomass would be essential for maturation of aerobic granules. Furthermore, it was found that the AI‐2‐associated coordination of microorganisms in microbial aggregates would be biomass density dependent. This study clearly shows the involvement of ATP and autoinducer‐2 in aerobic granulation, and may be exploited further for enhancement or prevention of microbial aggregation in general, for example, rapid granulation for wastewater treatment or inhibition of biofouling in membrane bioreactor. Biotechnol. Bioeng. 2010;105: 51–58. © 2009 Wiley Periodicals, Inc.     

State of the art of aerobic granulation in continuous flow bioreactors

In the wake of the success of aerobic granulation in sequential batch reactors (SBRs) for treating wastewater, attention is beginning to turn to continuous flow applications. This is a necessary step given the advantages of continuous flow treatment processes and the fact that the majority of full-scale wastewater treatment plants across the world are operated with aeration tanks and clarifiers in a continuous flow mode. As in SBRs, applying a selection pressure, based on differences in either settling velocity or the size of the biomass, is essential for successful granulation in continuous flow reactors (CFRs). CFRs employed for aerobic granulation come in multiple configurations, each with their own means of achieving such a selection pressure. Other factors, such as bioaugmentation and hydraulic shear force, also contribute to aerobic granulation to some extent. Besides the formation of aerobic granules, long-term stability of aerobic granules is also a critical issue to be addressed. Inorganic precipitation, special inocula, and various operational optimization strategies have been used to improve granule long-term structural integrity. Accumulated studies reviewed in this work demonstrate that aerobic granulation in CFRs is capable of removing a wide spectrum of contaminants and achieving properties generally comparable to those in SBRs. Despite the notable research progress made toward successful aerobic granulation in lab-scale CFRs, to the best of our knowledge, there are only three full-scale tests of the technique, two being seeded with anammox-supported aerobic granules and the other with conventional aerobic granules; two other process alternatives are currently in development. Application of settling- or size-based selection pressures and feast/famine conditions are especially difficult to implement to these and similar mainstream systems. Future research efforts needs to be focused on the optimization of the granule-to-floc ratio, enhancement of granule activity, improvement of long-term granule stability, and a better understanding of aerobic granulation mechanisms in CFRs, especially in full-scale applications.