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.     

Reactivation characteristics of stored aerobic granular sludge using different operational strategies

Aerobic granules after 6 months storage were employed in identical sequencing batch reactors (SBRs) using synthetic wastewater to investigate the impacts of different operational strategies on granules' reactivation process. The SBRs were operated under three operational strategies for reactivation of (a) different organic loading rate (OLR); (b) different ammonia concentration; and (c) different shear force (a superficial upflow air velocity). The results indicated that granules after long-term storage could be successfully recovered after 7 days of operation, and the excellent granule reactivation performance was closely related to the operational strategies, since inappropriate operational strategies could cause the outgrowth of filamentous bacteria and granule disintegration. Based on comprehensive comparison of reactivation performance under different operational strategies, the optimal operation strategy for granule reactivation was suggested at OLR of 0.8 kg COD/m(3)/day, ammonia concentration of 15-20 mg/L, and a superficial upflow air velocity of 2.6 cm/s. After 7 days operation under the optimal strategy, the dark brown granules (12 months storage) restored their bioactivities to previous state, in terms of COD removal efficiency (97.44%) and specific oxygen uptake rate (40.63 mg O(2)/g SS h(-1)). The results shed light on the future practical application of stored aerobic granules as bioseed for reactor fast start-up.     

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.     

Dependence of structure stability and integrity of aerobic granules on ATP and cell communication

Aerobic granules are dense and compact microbial aggregates with various bacterial species. Recently, aerobic granulation technology has been extensively explored for treatment of municipal and industrial wastewaters. However, little information is currently available with regard to their structure stability and integrity at levels of energy metabolism and cell communication. In the present study, a typical chemical uncoupler, 3,3′,4′,5-tetrachlorosalicylanilide with the power to dissipate proton motive force and subsequently inhibit adenosine triphosphate (ATP) generation, was used to investigate possible roles of ATP and cell communication in maintaining the structure stability and integrity of aerobic granules. It was found that inhibited ATP synthesis resulted in the reduced production of autoinducer-2 and N-acylhomoserine lactones essential for cell communication, while lowered extracellular polymeric substance (EPS) production was also observed. As a consequence, aerobic granules appeared to break up. This study showed that ATP-dependent quorum sensing and EPS were essential for sustaining the structure stability and integrity of aerobic granules.     

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.     

降解苯胺和氯苯胺类污染物好氧污泥颗粒化及微生物种群结构分析

以序批式气提生物反应器(SABR)为平台,研究了苯胺和氯苯胺类有毒有机废水处理过程好氧污泥颗粒化。结果表明,通过缩短污泥沉降时间、逐步提升目标污染物进水负荷,反应器连续运行3个月,最终在污泥沉降时间5min、COD负荷1.0~3.6kg/(m3.d)、苯胺和氯苯胺负荷1kg/(m3.d)条件下实现污泥颗粒化,COD、苯胺和氯苯胺去除率分别稳定在90%、99.9%以上;获得的成熟好氧颗粒粒径在0.45~2.5mm,SOUR稳定在150mgDO/(gVSS·h)以上,颗粒污泥EPS中PN含量为28.0±1.9mg/gVSS,PN/PS比值为6.5mg/mg,苯胺类比降解速率达0.18g/(g·d);应用PCR-DGGE分子指纹图谱技术分析了稳定运行的颗粒化反应器内好氧污泥微生物种群结构,结果表明好氧颗粒内主要细菌分属β-Proteobacteria、γ-Proteobacteria及Flavobacteria等类群,优势菌为Pseudomonas sp.、Flavobacterium sp.;与已获得的降解氯苯胺好氧颗粒相比,苯胺存在下培养获得的好氧颗粒污泥微生物菌群结构更为丰富。     

Respirometric Activities of Heterotrophic and Nitrifying Populations in Aerobic Granules Developed at Different Substrate N/COD Ratios

Aerobic granules were successfully developed at substrate N/COD ratios ranging from 5/100 to 30/100 by weight. By measuring respective respirometric activities of heterotrophic, ammonia-oxidizing, and nitrite-oxidizing bacteria, it was found that the relative abundance of nitrifying bacteria over heterotrophs in aerobic granules was closely related to the substrate N/COD ratios. Results further showed that the populations of both ammonia and nitrite oxidizers were significantly enriched with the increase of the substrate N/COD ratio, while a decreasing trend of heterotrophic population was observed in the aerobic granules. These seem to indicate that high substrate N/COD ratio favors the selection of nitrifying bacteria in the aerobic granules, while the relative activity of nitrifying population against heterotrophic population evolved until a balance between two populations was reached in the aerobic granular sludge community. Moreover, cell elemental composition was correlated with the shift in microbial populations, e.g., the enriched nitrifying population in the aerobic granules resulted in a high cell nitrogen content normalized to cell carbon content. This study provides a good insight into microbial interaction in aerobic granules.     

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.     

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.     

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.     

Evaluation on factors influencing the heterotrophic growth on the soluble microbial products of autotrophs

In this work, the heterotrophic growth on the microbial products of autotrophs and the effecting factors were evaluated with both experimental and modeling approaches. Fluorescence in situ hybridization (FISH) analysis illustrated that ammonia oxidizers (AOB), nitrite oxidizers (NOB), and heterotrophs accounted for about 65%, 20%, and 15% of the total bacteria, respectively. The mathematical evaluation of experimental data reported in literature indicated that heterotrophic growth in nitrifying biofilm (30–50%) and granules (30%) was significantly higher than that of nitrifying sludge (15%). It was found that low influent ammonium resulted in a lower availability of soluble microbial products (SMP) and a slower heterotrophic growth, but high ammonium (>150 mg N L⁻¹) feeding would lead to purely AOB dominated sludge with high biomass‐associated products contained effluent, although the absolute heterotrophic growth increased. Meanwhile, the total active biomass concentration increased gradually with the increasing solids retention time, whereas the factions of active AOB, NOB, and heterotrophs varied a lot at different solids retention times. This work could be useful for better understanding of the autotrophic wastewater treatment systems. Biotechnol. Bioeng. 2011; 108:804–812. © 2010 Wiley Periodicals, Inc.     

Diatom-derived carbohydrates as factors affecting bacterial community composition in estuarine sediments

Microphytobenthic biofilms in estuaries, dominated by epipelic diatoms, are sites of high primary productivity. These diatoms exude large quantities of extracellular polymeric substances (EPS) comprising polysaccharides and glycoproteins, providing a substantial pool of organic carbon available to heterotrophs within the sediment. In this study, sediment slurry microcosms were enriched with either colloidal carbohydrates or colloidal EPS (cEPS) or left unamended. Over 10 days, the fate of these carbohydrates and changes in beta-glucosidase activity were monitored. Terminal restriction fragment length polymorphism (T-RFLP), DNA sequencing, and quantitative PCR (Q-PCR) analysis of 16S rRNA sequences were used to determine whether sediment bacterial communities exhibited compositional shifts in response to the different available carbon sources. Initial heterotrophic activity led to reductions in carbohydrate concentrations in all three microcosms from day 0 to day 2, with some increases in beta-glucosidase activity. During this period, treatment-specific shifts in bacterial community composition were not observed. However, by days 4 and 10, the bacterial community in the cEPS-enriched sediment diverged from those in colloid-enriched and unamended sediments, with Q-PCR analysis showing elevated bacterial numbers in the cEPS-enriched sediment at day 4. Community shifts were attributed to changes in cEPS concentrations and increased beta-glucosidase activity. T-RFLP and sequencing analyses suggested that this shift was not due to a total community response but rather to large increases in the relative abundance of members of the gamma-proteobacteria, particularly Acinetobacter-related bacteria. These experiments suggest that taxon- and substrate-specific responses within the bacterial community are involved in the degradation of diatom-derived extracellular carbohydrates.     

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.     

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 μm into the granules leaving large anoxic zones in the centre part where denitrification can occur. In granules > 500 μm in diameter, Accumulibacter spp. was dominant in the outermost 200 μm 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.     

Diatom-Derived Carbohydrates as Factors Affecting Bacterial Community Composition in Estuarine Sediments

Microphytobenthic biofilms in estuaries, dominated by epipelic diatoms, are sites of high primary productivity. These diatoms exude large quantities of extracellular polymeric substances (EPS) comprising polysaccharides and glycoproteins, providing a substantial pool of organic carbon available to heterotrophs within the sediment. In this study, sediment slurry microcosms were enriched with either colloidal carbohydrates or colloidal EPS (cEPS) or left unamended. Over 10 days, the fate of these carbohydrates and changes in β-glucosidase activity were monitored. Terminal restriction fragment length polymorphism (T-RFLP), DNA sequencing, and quantitative PCR (Q-PCR) analysis of 16S rRNA sequences were used to determine whether sediment bacterial communities exhibited compositional shifts in response to the different available carbon sources. Initial heterotrophic activity led to reductions in carbohydrate concentrations in all three microcosms from day 0 to day 2, with some increases in β-glucosidase activity. During this period, treatment-specific shifts in bacterial community composition were not observed. However, by days 4 and 10, the bacterial community in the cEPS-enriched sediment diverged from those in colloid-enriched and unamended sediments, with Q-PCR analysis showing elevated bacterial numbers in the cEPS-enriched sediment at day 4. Community shifts were attributed to changes in cEPS concentrations and increased β-glucosidase activity. T-RFLP and sequencing analyses suggested that this shift was not due to a total community response but rather to large increases in the relative abundance of members of the γ-proteobacteria, particularly Acinetobacter-related bacteria. These experiments suggest that taxon- and substrate-specific responses within the bacterial community are involved in the degradation of diatom-derived extracellular carbohydrates.     

A comparative study on the formation and characterization of aerobic 4-chloroaniline-degrading granules in SBR and SABR

The formation and characterization of the aerobic 4-chloroaniline-degrading granules in the three column-type sequencing batch reactors were investigated in this paper. The granular sludge was observed since 15 days after start-up in R2 and R3 which had the high ratio of height to diameter (H/D). Since then and within the subsequent 75 days, the granulation of aerobic sludge was apparently developed by the decreased settling time and gradually increased 4-chloroaniline (4-ClA) concentration to above 400 mg.L(-1) in R1 to R3. The aerobic granules tended to be mature in all reactors continuously operated with 4-ClA loading rates of around 800 g.m(-3).d(-1), and the removal efficiencies of chemical oxygen demand, total nitrogen, and 4-ClA were maintained above 93%, 70%, and 99.9%, respectively. Mature aerobic granules in R1 to R3 featured with the average diameter of 0.78, 1.68, and 1.25 mm, minimal settling velocity of 20.5, 70.1 and 66.6 m.h(-1), specific 4-ClA degradation rates of 0.14, 0.21, and 0.27 g.gVSS(-1).d(-1), and the ratio of proteins to polysaccharides of 8.2, 10.8, and 13.7 mg.mg(-1), respectively. This study demonstrates that the reactor with a high H/D ratio and internal circulation favors the granulation and stabilization of aerobic sludge.     

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.     

Bacterial [methyl-3H]thymidine incorporation in substrate-amended estuarine sediment slurries

Abstract Five different bacterial communities were enriched in substrate-amended slurries of sediment from the Tay Estuary, Scotland. During incubation of the slurries, concentrations of volatile fatty acids, sulphate, sulphide and methane were monitored to clearly define the activity of the stimulated populations. An aerobic population, a ‘microaerophilic’ population and three anaerobic populations (fermentative heterotrophs, sulphate-reducing bacteria and methanogens plus acetogens) were established to reflect community growth and metabolism both in surface oxic and deeper anoxic layers. Similar numbers of cells involved in division were observed in all five slurries, demonstrating the potential for bacterial production. Thymidine incorporation rates in glucose-stimulated slurries under both aerobic and fully anaerobic conditions were similar, confirming the ability of fermentative anaerobic heterotrophs to incorporate [ methyl -³H]thymidine into DNA during growth. Although anaerobic communities of sulphate-reducing, acetogenic plus methanogenic bacteria were stimulated and actively growing, they did not incorporate [ methyl -³H]thymidine into DNA. Since the thymidine technique does not measure the growth of these important groups, calculated productivity values based upon thymidine incorporation within anoxic sediment systems will be substantially underestimated, even if growth substrates are not limiting.     

Heterotrophs grown on the soluble microbial products (SMP) released by autotrophs are responsible for the nitrogen loss in nitrifying granular sludge

In this work, nitrogen loss in the nitrite oxidation step of the nitrification process in an aerobic‐granule‐based reactor was characterized with both experimental and modeling approaches. Experimental results showed that soluble microbial products (SMP) were released from the nitrite‐oxidizing granules and were utilized as a carbon source by the heterotrophs for denitrification. This was verified by the fluorescence in situ hybridization (FISH) analysis. Microelectrode tests showed that oxygen diffusion limitation did result in an anoxic micro‐zone in the granules and allowed sequential utilization of nitrate as an electron acceptor for heterotrophic denitrification with SMP as a carbon source. To further elucidate the nitrogen loss mechanisms, a mathematic model was formulated to describe the growth of nitrite oxidizers, the formation and consumption of SMP, the anoxic heterotrophic growth on SMP and nitrate, as well as the oxygen transfer and the substrate diffusion in the granules. The results clearly indicate that the heterotrophs grown on the SMP released by the autotrophs are responsible for the nitrogen loss in the nitrifying granules, and give us a better understanding of the aerobic granules for nitrogen removal. Biotechnol. Bioeng. 2011;108: 2844–2852. © 2011 Wiley Periodicals, Inc.     

High organic loading influences the physical characteristics of aerobic sludge granules

Aims:  The effect of high organic loading rate (OLR) on the physical characteristics of aerobic granules was studied.
Methods and Results:  Two column-type sequential aerobic sludge blanket reactors were fed with either glucose or acetate as the main carbon source, and the OLR was gradually raised from 6 to 9, 12 and 15 kg chemical oxygen demand (COD) m⁻³ d⁻¹. Glucose-fed granules could sustain the maximum OLR tested. At a low OLR, these granules exhibited a loose fluffy morphology dominated by filamentous bacteria. At higher OLRs, these granules became irregularly shaped, with folds, crevices and depressions. In contrast, acetate-fed granules had a compact spherical morphology at OLRs of 6 and 9 kg COD m⁻³ d⁻¹, with better settling and strength characteristics than glucose-fed granules at similar OLRs. However, acetate-fed granules could not sustain high OLRs and disintegrated when the OLR reached 9 kg COD m⁻³ d⁻¹.
Conclusions:  The compact regular microstructure of the acetate-fed granules appeared to limit mass transfer of nutrients at an OLR of 9 kg COD m⁻³ d⁻¹. The looser filamentous microstructure of the glucose-fed granules and the subsequent irregular morphology delayed the onset of diffusion limitation and allowed significantly higher OLRs to be attained.
Significance and Impact of the Study:  High organic loading rates are possible with aerobic granules. This research would be helpful in the development of aerobic granule-based systems for high-strength wastewaters.