Organics and nitrogen removal and sludge stability in aerobic granular sludge membrane bioreactor

Novel aerobic granular sludge membrane bioreactor (GMBR) was established by combining aerobic granular sludge technology with membrane bioreactor (MBR). GMBR showed good organics removal and simultaneous nitrification and denitrification (SND) performances for synthesized wastewater. When influent total organic carbon (TOC) was 56.8-132.6 mg/L, the TOC removal of GMBR was 84.7-91.9%. When influent ammonia nitrogen was 28.1-38.4 mg/L, the ammonia nitrogen removal was 85.4-99.7%, and the total nitrogen removal was 41.7-78.4%. Moreover, batch experiments of sludge with different particle size demonstrated that: (1) flocculent sludge under aerobic condition almost have no denitrification capacity, (2) SND capacity was caused by the granular sludge, and (3) the denitrification rate and total nitrogen removal efficiency were enhanced with the increased particle size. In addition, study on the sludge morphology stability in GMBR showed that, although some granular sludge larger than 0.9 mm disaggregated at the beginning of operation, the granular sludge was able to maintain the stability of its granular morphology, and at the end of operation, the amount of granular sludge (larger than 0.18 mm) stabilized in GMBR was more than 56-62% of the total sludge concentration. The partial disaggregation of large granules is closely associated with the change of operating mode from sequencing batch reactor (SBR) system to MBR system.     

Enhanced biological phosphate removal by granular sludge in a sequencing batch reactor

A laboratory-scale sequencing batch reactor was started-up with flocculated biomass and operated primarily for enhanced biological phosphate removal. Ten weeks after the start-up, gradual formation of granular sludge was observed. The compact biomass structure allowed halving the settling time, the initial reactor volume, and doubling the influent COD concentration. Continued operation confirmed the possibility of maintaining a stable granular biomass with a sludge volume index less than 40 ml g^–1, while securing a removal efficiency of 95% for carbon, 99.6% for phosphate, and 71% for nitrogen. Microscopic observations revealed a morphological diversity.     

Isolation of hydrogen-producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor

H₂-producing bacteria were isolated from anaerobic granular sludge. Out of 72 colonies (36 grown under aerobic conditions and 36 under anaerobic conditions) arbitrarily chosen from the agar plate cultures of a suspended sludge, 34 colonies (15 under aerobic conditions and 19 under anaerobic conditions) produced H₂ under anaerobic conditions. Based on various biochemical tests and microscopic observations, they were classified into 13 groups and tentatively identified as follows: From aerobic isolates,Aeromonas spp. (7 strains),Pseudomonas spp. (3 strains), andVibrio spp. (5 strains); from anaerobic isolates,Actinomyces spp. (11 strains),Clostridium spp. (7 strains), andPorphyromonas sp. When glucose was used as the carbon substrate, all isolates showed a similar cell density and a H₂ production yield in the batch cultivations after 12h (2.24–2.74 OD at 600 nm and 1.02–1.22 mol H₂/mol glucose, respectively). The major fermentation by-products were ethanol and acetate for the aerobic isolates, and ethanol, acetate and propionate for the anaerobic isolates. This study demonstrated that several H₂ producers in an anaerobic granular sludge exist in large proportions and their performance in terms of H₂ production is quite similar.     

Effects of ferrous iron on the performance and microbial community in aerobic granular sludge in relation to nutrient removal

Lab‐scale experiments were conducted to investigate the effects of ferrous iron on nutrient removal performance and variations in the microbial community inside aerobic granular sludge for 408 days. Two reactors were simultaneously operated, one without added ferrous iron (SBR1), and one with 10 mg Fe²⁺ L^?1 of added ferrous iron (SBR2). A total of 1 mg Fe²⁺ L^?1 of added ferrous iron was applied to SBR1 starting from the 191st day to observe the resulting variations in the nutrient removal performance and the microbial community. The results show that ammonia‐oxidizing bacteria (AOB) could not oxidize ammonia due to a lack of iron compounds, but they could survive in the aerobic granular sludge. Limited ferrous iron addition encouraged nitrification. Enhanced biological phosphorus removal (EBPR) from both reactors could not be maintained regardless of the amount of ferrous iron that was applied. EBPR was established in both reactors when the concentration of mixed liquor suspended solid (MLSS) and the percentage of Accumulibacteria increased. A total of 10 mg Fe²⁺ L^?1 of added ferrous iron had a relatively adverse effect on the growth of AOB species compared to 1 mg Fe²⁺ L^?1 of added ferrous iron, but it encouraged the growth of Nitrospira sp. and Accumulibacteria, which requires further study. It could be said that the compact and stable structure of aerobic granular sludge preserved AOB and NOB from Fe‐deficient conditions, and wash‐out during the disintegration period. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:716–725, 2017     

好氧颗粒污泥对不同底物组成的响应

在序批式间歇反应器（R1、R2和R3）中,采用乙酸钠（R1）、蔗糖（R2）和苯酚（R3）三种不同基质作为碳源,均成功地培养出了好氧颗粒污泥;考察了不同颗粒污泥的理化性质及其对污染物的转化能力。结果表明,R1中颗粒污泥外观呈黄色,其主要的微生物菌群为细菌;R2中颗粒污泥外观呈黑色,内部含有丝状菌;而R3中颗粒污泥表面被大量丝状菌包裹,颗粒污泥呈淡黄色。在进水COD1000mg／L时R1、R2和R3中颗粒污泥比有机物的利用速率大小顺序为R3〉R1〉R2,而COD的去除率顺序却为R2〉R1〉R3。在进水氨氮40mg／L时,R1、R2和R3中氨氮的去除率分别在91％、96％和80％以上。以不同的底物培养出不同的好氧颗粒污泥可以拓展其在有毒化学物质如酚类化合物和高浓度工业废水生物处理中的应用。     

Aerobic granular sludge: characterization,mechanism of granulation and application to wastewater treatment

Aerobic granular sludge can be classified as a type of self-immobilized microbial consortium, consisting mainly of aerobic and facultative bacteria and is distinct from anaerobic granular methanogenic sludge. Aerobic granular technology has been proposed as a promising technology for wastewater treatment, but is not yet established as a large-scale application. Aerobic granules have been cultured mainly in sequenced batch reactors (SBR) under hydraulic selection pressure. The factors influencing aerobic granulation, granulation mechanisms, microbial communities and the potential applications for the treatment of various wastewaters have been studied comprehensively on the laboratory-scale. Aerobic granular sludge has shown a potential for nitrogen removal, but is less competitive for the high strength organic wastewater treatments. This technology has been developed from the laboratory-scale to pilot scale applications, but with limited and unpublished full-scale applications for municipal wastewater treatment. The future needs and limitations for aerobic granular technology are discussed.     

Cobalt toxicity in anaerobic granular sludge: influence of chemical speciation

The influence of cobalt speciation on the toxicity of cobalt to methylotrophic methanogenesis in anaerobic granular sludge was investigated. The cobalt speciation was studied with three different media that contained varying concentrations of complexing ligands [carbonates, phosphates and ethylenediaminetetraacetic acid (EDTA)]. Three fractions (nominal added, dissolved and free) of cobalt were determined in the liquid media and were correlated with data from batch toxicity experiments. The average concentration of cobalt that was required for 50% inhibition of methanogenic activity (IC₅₀) for free Co²⁺ in the three sets of measurements was 13 μmol/L with a standard deviation of 22% and a similarity of 72% between the data obtained in the three different media for the range of cobalt concentrations investigated. The standard deviation of the IC₅₀ for the other two fractions was much higher, i.e. 85 and 144% for the added cobalt and dissolved cobalt, respectively, and the similarity was almost 0% for both fractions. Complexation (and precipitation) with EDTA, phosphates and carbonates was shown to decrease the toxicity of cobalt on methylotrophic methanogenesis. The free cobalt concentration is proposed to be the key parameter to correlate with cobalt toxicity. Thus, the toxicity of cobalt to granular sludge can be estimated based on the equilibrium-free cobalt concentration.     

Methanogenic granular sludge formation in an upflow anaerobic sludge blanket reactir treating synthetic methanolic waste

A laboratory upflow anaerobic sludge blanket reactor, seeded with fine, suspended, bacterial floc with 1.76 g volatile suspended solids/l, was used to treat synthetic methanolic waste. After 180 days of continuous peration, granular sludge with discrete granules of 1 to 2 mm diam. was formed, with 52 g volatile suspended solids/l. Granules were brown, relatively soft and had a settling velocity of 1.61 cm/s. Extracellular polymeric matter extracted from the granular sludge had high carbohydrate content but low nucleic acid content. The ash of the granular sludge contained Na⁺, K⁺ and Mg²⁺ up to 15.0, 11.7 and 3.75 mg/g, respectively. Scanning and transmission electron microscopy revealed that the granular sludge was dominated by methanogens resembling Methanosarcina.The authors are with the Department of Environmental Engineering, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan     

Start-up and inhibition analysis of the Anammox process seeded with anaerobic granular sludge

The longer start-up period of the Anammox process is due to the very low cellular yield and growth rates of Anammox bacteria. Nitrite inhibition is considered to be the key factor in the instability of the Anammox process during the operation. However, little attention was paid to the inhibitory effect of pH and free ammonia. This paper presents start-up and inhibition analysis of an Anammox biofilm reactor seeded with anaerobic granular sludge. Results showed that the start-up period could be divided into the sludge lysis phase, lag phase, propagation phase, stationary phase and inhibition phase. Optimization control could be implemented correspondingly to accelerate the start-up of Anammox bioreactors. Effluent pH increased to 8.7–9.1 when the nitrogen removal rate was higher than 1,200 mg l⁻¹ day⁻¹. The free ammonia concentration was accompanied with a higher level of 64–73 mg l⁻¹. Inhibitory effects of high pH and free ammonia on Anammox bacteria contributed to the destabilization of the Anammox bioreactor during the first 125 days with influent KHCO₃ of 0.5 g l⁻¹. Increasing the suffering capacity in the inlet by dosing 1.25 g KHCO₃ l⁻¹ effectively reduced the pH variation, and the nitrogen removal performance of the reactor was further developed.     

Influence of pH shocks on trace metal dynamics and performance of methanol fed granular sludge bioreactors

The influence of pH shocks on the trace metal dynamics and performance of methanol fed upflow anaerobic granular sludge bed (UASB) reactors was investigated. For this purpose, two UASB reactors were operated with metal pre-loaded granular sludge (1mM Co, Ni and Fe; 30°C; 96h) at an organic loading rate (OLR) of 5gCOD l reactor^–1d^–1. One UASB reactor (R1) was inoculated with sludge that originated from a full scale reactor treating alcohol distillery wastewater, while the other reactor (R2) was inoculated with sludge from a full scale reactor treating paper mill wastewater. A 30h pH shock (pH 5) strongly affected the metal retention dynamics within the granular sludge bed in both reactors. Iron losses in soluble form with the effluent were considerable: 2.3 and 2.9% for R1 and R2, respectively, based on initial iron content in the reactors, while losses of cobalt and nickel in soluble form were limited. Sequential extraction of the metals from the sludge showed that cobalt, nickel, iron and sulfur were translocated from the residual to the organic/sulfide fraction during the pH shock in R2, increasing 34, 47, 109 and 41% in the organic/sulfide fraction, respectively. This is likely due to the modification of the iron sulfide precipitate stability, which influences the extractability of iron and trace metals. Such a translocation was not observed for the R1 sludge during the first 30h pH shock, but a second 4day pH shock induced significant losses of cobalt (18%), iron (29%) and sulfur (29%) from the organic/sulfide fraction, likely due to iron sulfide dissolution and concomitant release of cobalt. After the 30h pH shock, VFA accumulated in the R2 effluent, whereas both VFA and methanol accumulated in R1 after the 4day pH shock. The formed VFA, mainly acetate, were not converted to methane due to the loss of methanogenic activity of the sludge on acetate. The VFA accumulation gradually disappeared, which is likely to be related to out-competition of acetogens by methanogens. Zinc, copper and manganese supply did not have a clear effect on the acetate removal and methanol conversion, but zinc may have induced the onset of methanol degradation after day 152 in R1.     

Feasibility of expanded granular sludge bed reactors for the anaerobic treatment of low-strength soluble wastewaters

The application of the expanded granular sludge bed (EGSB) reactor for the anaerobic treatment of low-strength soluble wastewaters using ethanol as a model substrate was investigated in laboratory-scale reactors at 30oC. Chemical oxygen demand (COD) removal efficiency was above 80% at organic loading rates up to12 g COD/L . d with influent concentrations as low as 100 to 200 mg COD/L. These results demonstrate the suitability of the EGBS reactor for the anaerobic treatment of low-strength wastewaters. The high treatment performance can be attributed to the intense mixing regime obtained by high hydraulic and organic loads. Good mixing of the bulk liquid phase for the substrate-biomass contact and adequate expansion of the substrate-biomass contact and adequate expansion of the sludge bed for the degassing were obtained when the liquid upflow velocity (V(up)) was greater than 2.5 m/h. Under such conditions, an extremely low apparent K(s) value for acetoclastic methanogenesis of 9.8 mg COD/L was observed. The presence of dissolved oxygen in the wastewater had no detrimental effect on the treatment performance. Sludge piston flotation from pockets of biogas accumulating under the sludge bed occurred at V(up) lower than 2.5 m/h due to poor bed expansion. This problem is expected only in small diameter laboratory-scale reactors. A. more important restriction of the EGSB reactor was the sludge washout occurring at V(up) higher than 5.5 m/h and which was intensified at organic loads higher than 7 g COD/L. d due to buoyancy forces from the gas production. To achieve an equilibrium between the mixing intensity and the sludge hold-up, the operation should be limited to an organic loading rate of 7 g COD/L d. and to a liquid up-flow velocity between 2.5 and 5.5 m/h (c) 1994 John Wiley & Sons, Inc.     

Enhancement of sulfate reduction activity using granular sludge in anaerobic treatment of acid mine drainage

Three laboratory-scale, upflow anaerobic reactors were operated for about 250 d to determine the effect of activated granular sludge with high density of sulfate reducing bacteria in the treatment of artificial acid mine drainage. Sulfate reducing bacteria in the granular sludge taken from the upflow anaerobic sludge blanket reactor were 1–2×10⁶ c.f.u. g^–1, which is at least 10 times higher than that of organic substrates such as cow manure and oak compost. The reactors with granular sludge effectively removed over 99% of heavy metals, such as Fe, Al, Cu, and Cd during the experiment. This result suggests a feasibility of the application of granular sludge as a source of sulfate reducing bacteria for the treatment of acid mine drainage.     

Monitoring of specific methanogenic activity of granular sludge by confocal laser scanning microscopy during start-up of thermophilic upflow anaerobic sludge blanket reactor

Confocal, laser-scanning microscopy was applied to acquire coenzyme F₄₂₀-based autofluorescence images of middle sections of sludge granules during start-up of a thermophilic reactor that were seeded with mesophilically-grown microorganisms of granular sludge. Digital images were analyzed to calculate weighted averages of autofluorescence. The values were related (r ²=0.97) to specific methanogenic activities of granular sludge as the granules developed to steady state.     

The settling characteristics and mean settling velocity of granular sludge in upflow anaerobic sludge blanket (UASB)-like reactors

Mean settling velocity of granular sludge in full-scale UASB (upflow anaerobic sludge blanket) and EGSB (expanded granular sludge bed) reactors was evaluated by settling column tests, and a settling velocity model based on the experimental results and available literature data was developed. It is concluded that the settling velocity should be calculated by the Allen formula, because the settling process of the granules is in the category of intermediate flow regime rather than in the laminar flow one. The comparison between calculated and measured values of the settling velocity shows an excellent agreement, with an average relative error of 4.04%. A simple but reliable mathematical method to determine the settling velocity is therefore proposed.     

The effect of electrodialytic treatment and Na2H2EDTA addition on methanogenic activity of copper-amended anaerobic granular sludge: treatment costs and energy consumption

The effect of electrodialytic treatment in terms of a current density, pH and Na₂H₂EDTA addition on the methanogenic activity of copper-amended anaerobic granular sludge taken from the UASB reactor from paper mill was evaluated. Moreover, the specific energy consumption and simplified operational and treatment costs were calculated. Addition of Na₂H₂EDTA (at pH 7.7) to copper-amended sludge resulted in the highest microbial activity (62 mg CH₄-COD g VSS⁻¹ day⁻¹) suggesting that Na₂H₂EDTA decreased the toxic effects of copper on the methanogenic activity of the anaerobic granular sludge. The highest methane production (159 %) was also observed upon Na₂H₂EDTA addition and simultaneous electricity application (pH 7.7). The energy consumption during the treatment was 560, 840, 1400 and 1680 kW h m⁻³ at current densities of 0.23, 0.34, 0.57 and 0.69 mA cm⁻², respectively. This corresponded to a treatment costs in terms of electricity expenditure from 39.2 to 117.6 € per cubic meter of sludge.     

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.     

Aerobic granular sludge formation for high strength agro-based wastewater treatment

The present study investigates the formation of aerobic granular sludge in sequencing batch reactor (SBR) fed with palm oil mill effluent (POME). Stable granules were observed in the reactor with diameters between 2.0 and 4.0 mm at a chemical oxygen demand (COD) loading rate of 2.5 kg COD m⁻³ d⁻¹. The biomass concentration was 7600 mg L⁻¹ while the sludge volume index (SVI) was 31.3 mL g SS⁻¹ indicating good biomass accumulation in the reactor and good settling properties of granular sludge, respectively. COD and ammonia removals were achieved at a maximum of 91.1% and 97.6%, respectively while color removal averaged at only 38%. This study provides insights on the development and the capabilities of aerobic granular sludge in POME treatment.     

Bacterial community dynamics in long‐term operation of a pilot plant using aerobic granular sludge to treat pig slurry

Aerobic granular sludge represents an interesting approach for simultaneous organic matter and nitrogen removal in wastewater treatment plants. However, the information about microbial communities in aerobic granular systems dealing with industrial wastewater like pig slurry is limited. Herein, bacterial diversity and dynamics were assessed in a pilot scale plant using aerobic granular sludge for organic matter and nitrogen elimination from swine slurry during more than 300 days. Results indicated that bacterial composition evolved throughout the operational period from flocculent activated sludge, used as inoculum, to mature aerobic granules. Bacterial diversity increased at the beginning of the granulation process and then declined due to the application of transient organic matter and nitrogen loads. The operational conditions of the pilot plant and the degree of granulation determined the microbial community of the aerobic granules. Brachymonas, Zoogloea and Thauera were attributed with structural function as they are able to produce extracellular polymeric substances to maintain the granular structure. Nitrogen removal was justified by partial nitrification (Nitrosomonas) and denitrification (Thauera and Zoogloea), while Comamonas was identified as the main organic matter oxidizing bacteria. Overall, clear links between bacterial dynamics and composition with process performance were found and will help to predict their biological functions in wastewater ecosystems improving the future control of the process. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1212–1221, 2016