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.     

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.     

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.     

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 μm 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.Stephen Tiong-Lee Tay Passed away on 27 July 2005.     

Degrading high-strength phenol using aerobic granular sludge

Aerobic granules were adopted to degrade high-strength phenol wastewater in batch experiments. The acclimated granules effectively degraded phenol at a concentration of up to 5,000 mg l⁻¹ without severe inhibitory effects. The biodegradation of phenol by activated sludge was inhibited at phenol concentrations >3,000 mg l⁻¹. The granules were composed of cells embedded in a compact extracellular matrix. After acid or alkaline pretreatment, the granules continued to degrade phenol at an acceptable rate. The polymerase chain reaction-denaturing gradient gel electrophoresis technique was employed to monitor the microbial communities of the activated sludge and the aerobic granules following their being used to treat high concentrations of phenol in batch tests.     

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.     

Influence of phenol on cultures of acetate-fed aerobic granular sludge

AIMS: This paper attempts to investigate the inhibition of phenol on the acetate utilization in acetate-fed aerobic granular sludge culture. METHODS AND RESULTS: Acetate-fed aerobic granules with a mean diameter of 1.0 mm were predeveloped in a column sequencing aerobic sludge blanket reactor. The present study looked into the utilization kinetics of acetate by acetate-fed aerobic granules in the presence of different phenol concentrations ranging from 0 mg l(-1) to 50 mg l(-1). For this purpose, batch experiments were conducted at 25 degrees C, while the initial biomass and acetate concentrations were in a range of 109-186 mg mixed liquor suspended solids (MLSS) l(-1) and 185-300 mg acetate-chemical oxygen demand (COD) l(-1). Results showed that the utilization of acetate in the presence of phenol was subject to a zero-order reaction kinetics. The relative phenol concentration in terms of the ratio of initial phenol concentration (C(p)) to initial biomass concentration (X(0)) was used to describe the real inhibitory strength of phenol imposed on acetate-fed aerobic granules. When the C(p)/X(0) ratio increased from 0 to 0.19 mg phenol mg(-1) MLSS, the zero-order reaction rate constant of acetate dropped from 1.15 mg l(-1) min(-1) to 0.38 mg l(-1) min(-1), and a similar trend was also observed in specific oxygen utilization rate. As compared to the control test without addition of phenol, the acetate-COD removal efficiency was reduced by nearly 50% at a C(p)/X(0) value of 0.19 mg phenol mg(-1) MLSS. It was found that biodegradation of phenol was negligible in acetate-fed aerobic granular sludge batch culture. CONCLUSIONS: It appears that phenol can seriously repress the utilization of acetate in the acetate-fed aerobic granular sludge batch cultures. A simple zero-order reaction model could adequately describe the utilization of acetate by acetate-fed aerobic granules in the presence of phenol. SIGNIFICANCE AND IMPACT OF THE STUDY: It is expected that this study would lead to a better understanding of the behaviour of acetate-fed aerobic granules in the presence of inhibitory organic compounds.     

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.     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Formation of aerobic granules and their PHB production at various substrate and ammonium concentrations

Aerobic granular sludge rich in polyhydroxybutyrate (PHB) was cultivated in a sequencing batch reactor (SBR) by seeding anaerobic granular sludge. The PHB content in aerobic granules was investigated and the experimental results reveal that both influent chemical oxygen demand (COD) and ammonium concentrations had a significant effect on the morphological characteristics and the PHB production of the aerobic granular sludge. At a COD and ammonium concentration of 750 mg/L and 8.5mg/L, respectively, the PHB content of the granules reached 44%, but their poor settling ability, as evidenced by a high sludge volume index, was observed. This was attributed to the outgrowth of filamentous bacteria on the granule surface. However, an increase in the ammonium concentration resulted in an elevated sludge concentration and a decrease in the PHB content in the granules. In this case, the aerobic granular sludge with a regular and compact structure was formed. The results suggest that, through controlling the COD and ammonium concentrations in the influent, the PHB-rich aerobic granular sludge with good settling ability could be cultivated.     

Aerobic granulation in sequencing batch reactors at different settling times

The present investigation examines the way to enhance aerobic granulation by controlling the microbial communities via applying different settling times. Early granulation of aerobic granules is noticeable at a settling time of 5 min. The functional strains are enriched in granules without challenge of non-flocculating strains. Short settling times at initial stage principally determine the efficiency of subsequent granulation processes.     

Biodegradation of dimethyl phthalate by Sphingomonas sp. isolated from phthalic-acid-degrading aerobic granules

This study isolated nine strains of aerobic phenol-degrading granules. These isolates (I1–I9) were characterized using 16S rRNA gene sequencing, with γ-Proteobacteria as the dominant strains in the aerobic granules. While most strains demonstrated either high phenol-degrading capabilities or auto-aggregation capabilities, three isolates, I2, I6, and I8 showed both features. These findings contradict the previous view that auto-aggregation and phenol degradation are mutually exclusive in aerobic granules. Strains I2 and I8 independently formed single-culture aerobic granules except for I3. Anti-microbial activity test results indicated that strains I2 and I8 inhibited growth of strain I3. However, co-culturing I3 with I2 or I8 helped to form granules.     

The effect of bioaugmentation on the performance of sequencing batch reactor and sludge characteristics in the treatment process of papermaking wastewater

In this paper, the differences between reinforced sequencing batch reactor, which was inoculated with superior mixed flora, and conventional sequencing batch reactor were compared in the process of treating papermaking wastewater under similar conditions. The results showed that the addition of superior mixed flora could not only shorten the sludge acclimation time, but also improve the treatment efficiency of reactor as well as make the reactor have higher ability to withstand high volume loading rate; the phenomenon of aerobic granulation only occurred in reinforced sequencing batch reactor, and superior mixed flora were the key reason that aerobic granular sludge could shape; aerobic granular sludge had many advantages over conventional activated sludge such as it possessed compacter microbial structure, better settling performance, and lower water content.     

A generalized model for settling velocity of aerobic granular sludge

Aerobic granulation is a novel biotechnology recently receiving intensive research attention. Aerobic granules developed in SBR can be as big as several millimeters, thus the traditional models describing the settling velocity of activated sludge are no long valid in aerobic granules culture. In this study, a new type of model was developed for the settling velocity of aerobic granules. This model shows that the settling velocity of aerobic granules is the function of SVI, mean size of granules and biomass concentration of granules. When the size of bioparticle is small enough, the proposed model reduces to the well-known Vesilind equation. Results indicated that the proposed model could satisfactorily fit experimental results obtained in the course of aerobic granulation under different conditions, while the Vesilind equation failed to or very poorly fit the experimental data. In addition, the proposed model can also be extended to anaerobic granules. The settling velocity is one of the most important parameters in both aerobic and anaerobic granulation, and successful biogranulation is highly related to the manipulation of settling velocity. It was demonstrated that the proposed model can sever as a useful tool for design and operation engineers to properly select the settling velocity for enhanced aerobic and anaerobic granulation.     

A unified theory for upscaling aerobic granular sludge sequencing batch reactors

Aerobic granular sludge sequencing batch reactors (SBR) are a promising technology for treating wastewater. Increasing evidence suggests that aerobic granulation in SBRs is driven by selection pressures exerted on microorganisms. Three major selection pressures have been identified as follows: settling time, volume exchange ratio and discharge time. This review demonstrates that these three major selection pressures can all be unified to one, the minimal settling velocity of bio-particles, that determines aerobic granulation in SBRs. The unified selection pressure theory is a useful guide for manipulating and optimizing the formation and characteristics of aerobic granules in SBRs. Furthermore, the unified theory provides a single engineering basis for scale up of aerobic granular sludge SBRs.     

Variable aeration in sequencing batch reactor with aerobic granular sludge

This study investigated the effects of reduced aeration in famine period on the performance of sequencing batch reactor (SBR) with aerobic granular sludge. Aerobic granules were first cultivated in two SBRs (R1 and R2) with acetate as sole carbon source. From operation day 27, aeration rate in R1 was reduced from 1.66 to 0.55 cm s(-1) from 110 min to the end of each cycle and further reduced from 30 min to the end of each cycle from day 63. R2 as a control was operated with a constant aeration rate of 1.66 cm s(-1) in the whole cycle during the entire experimental period. Results showed that changing trends of SVI, concentration, average size and VSS/SS of biomass with time in R1 and R2 were similar although different aeration modes were adopted. At steady state, SVI of aerobic granules and biomass concentration maintained at about 40 ml g(-1) and 6 g l(-1), respectively. Average size of granules was about 750 microm in R1 while 550 microm in R2. This is the first study to demonstrate that aerobic granular sludge could be stable at reduced aeration rate in famine period during more than 3-month operation. Such an operation strategy with reduced aeration rate will lead to a significant reduction of energy consumption, which makes the aerobic granular sludge technology more competitive over conventional activated sludge process. Furthermore, the stability of aerobic granular system with variable aeration further indicates that the difference of physiology and kinetics of aerobic granule in feast and famine periods results in the different requirements of oxygen and shear stress for the stability of granules, which will deepen the understanding of mechanism of aerobic granulation in sequencing batch reactor.     

Changes in structure,activity and metabolism of aerobic granules as a microbial response to high phenol loading

Four column-type sequential aerobic sludge blanket reactors were fed with phenol as the sole carbon and energy source and operated at loading rates of 1.0, 1.5, 2.0 and 2.5 kg phenol m^–3 day^–1. The results indicated that phenol loading exerted a profound influence on the structure, activity and metabolism of the aerobic granules. Compact granules with good settling ability were maintained at loadings up to 2.0 kg phenol m^–3 day^–1, and structurally weakened granules with enhanced production of extracellular polymers and proteins and significantly lower hydrophobicities were observed at the highest loading of 2.5 kg phenol m^–3 day^–1. Specific oxygen uptake rate, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) activities peaked at a loading of 2.0 kg phenol m^–3 day^–1, and declined thereafter. Granules degraded phenol completely in all four reactors, mainly through the meta cleavage pathway as C23O activities were significantly higher than C12O activities. At the highest loading applied, the anabolism and catabolism of microorganisms were regulated such that phenol degradation proceeded exclusively via the meta pathway, apparently to produce more energy for overstimulation of protein production against phenol toxicity. This work contributes to a better understanding of the ability of aerobic granules to handle high-strength industrial wastewaters containing chemicals that are normally inhibitory to microbial growth.     

两株高效苯酚降解菌的筛选、鉴定及生物强化-DTRO组合工艺初步验证

【目的】从煤化工废水中分离、筛选苯酚高效降解微生物,初步考察微生物与DTRO技术联用,构建含酚废水生物强化处理工艺的可行性。【方法】采用苯酚浓度梯度培养基对苯酚降解微生物进行分离和筛选;根据菌体形态电子显微镜观察、菌株生理生化特性考察和16S r RNA基因系统发育树构建,对菌株进行初步生物学鉴定;将筛选出的高效苯酚降解菌制备成相应的菌剂与碟管式反渗透(DTRO)技术组合形成"生物强化-DTRO"工艺,并试用于含酚废水的处理。【结果】共获得7株纯化细菌,其中Phe-03和Phe-05为高效苯酚降解菌;该2株菌均可以苯酚为唯一碳源生长。经鉴定Phe-03为壤霉菌属(Agromyces)菌株;Phe-05为棒杆菌属(Corynebacterium)菌株。到目前为止,壤霉菌属(Agromyces)菌株降解苯酚尚未见报道。在初始苯酚浓度达到1 300 mg/L条件下,Phe-03和Phe-05菌株44 h内对苯酚降解率均达到70%以上;76 h后苯酚降解率均超过90%。组合形成的"生物强化-DTRO"工艺不仅可以有效去除废水中的酚类化合物,而且还能减少反渗透膜污染,以及增加膜的通透性。【结论】研究表明微生物技术可与DTRO技术联用,构建含酚废水生物强化处理工艺,可为含酚废水处理技术研究提供一种选择思路。     

Stoichiometric analysis of dissolved organic carbon flux into storage and growth in aerobic granules culture

This study attempted to address a fundamental question of whether metabolic behaviors of aerobic granules are different from their counterparts, such as activated sludge and biofilms. A series of respirometric experiments were carried out using mature aerobic granules with mean sizes of 0.75–3.4 mm. Results suggested that metabolism of aerobic granules comprised three consecutive phases: (i) conversion of external dissolved organic carbon to a poly-β-hydroxybutyrate-like substance; (ii) growth of aerobic granules on the stored poly-β-hydroxybutyrate-like substance derived from phase I, and (iii) subsequent endogenous metabolism of aerobic granules. The stoichiometric analysis revealed that the conversion yields of external dissolved organic carbon to the poly-β-hydroxybutyrate-like substance, the growth yields of biomass on storage, and the overall growth yields of biomass on external dissolved organic carbon were not significantly correlated to the sizes of aerobic granules, i.e., the metabolism of aerobic granules would be size independent. The conversion coefficients and growth rates of aerobic granules were found to be comparable with those reported in the activated sludge and biofilms cultures, indicating that there would not be significant difference in the metabolisms of aerobic granules over activated sludge and biofilms. This information will be useful for modeling and designing aerobic granular sludge processes.