Microbial Granules Research Articles

Aerobic granulation for organic carbon and nitrogen removal in alternating aerobic–anaerobic sequencing batch reactor

Microbial granules with excellent settleability were successfully developed at different ammonium–N loadings in four sequencing batch reactors run under alternating aerobic–anaerobic conditions. Results showed that heterotrophic, nitrifying and denitrifying populations could co-exist in the microbial granules. The activity of denitrifying bacteria was highly determined by the availability of external carbon source in the anaerobic phase. Complete denitrification was achieved with supply of external carbon source, whereas only partial denitrification occurred with no addition of external carbon source. This study shows that microbial granules cultivated under the alternating aerobic–anaerobic condition could efficiently remove organic carbon and completely convert ammonia to nitrogen gas. A new generation of microbial granules-based bioreactor for organics and nitrogen removal can be envisaged.

Formation and structure of granulated microbial aggregates used in aerobic wastewater treatment

Granular microbial aggregates are used in aerobic treatment of wastewater. The granules have diverse microbial community and complex spatial structure. The structural elements are radial sub-aggregates, concentric layers, channels, pores, polysaccharide plugs, and an anaerobic core of lysed cells. Aerobic bacteria, consisting of 69–84% of microbial biomass, were concentrated in a layer to the depth of 550 μm from the surface of the granule. Facultative anaerobic bacteria, consisting of 9–13% of microbial biomass, dominated in a layer at a depth from 550 μm to 850 μm from the surface of the granule. Obligate anaerobic bacteria, consisting of 2% of microbial biomass, dominated in a layer on the depth from 850 μm to 1,000 μm from the surface of the granule. A core of dead and lysed cells was at a depth greater than 1,000 μm from the surface of the granule. The depth of the anaerobic layer correlated with the appearance of polysaccharide plugs in the pores. Enrichment cultures of microorganisms with high cell surface hydrophobicity or self-aggregation ability can be used to facilitate the formation of microbial granules.

Influence of phenol on nitrification by microbial granules

The effect of phenol on the nitrifying activity of aerobically grown microbial granules, produced in a sequencing batch reactor, was evaluated in batch experiments. Ammonium oxidation rate and nitrate production rate decreased in the presence of phenol. Nitrifying activity was recovered completely after degradation of phenol when initial phenol concentrations were not higher than 10 mg l −1. For initial phenol concentrations of 15 and 20 mg l −1, the recoveries of nitrate productivity were 77% and 67% of the control, respectively. The results showed that nitrifying bacteria embedded in microbial granules were relatively resilient to the inhibitory effects exerted by phenol. These aerobic microbial granules could potentially be applied to simultaneous removal of organics and ammonia from wastewaters containing phenol.

The elemental compositions of P-accumulating microbial granules developed in sequencing batch reactors

The effects of substrate P/COD ratios on the elemental compositions of P-accumulating microbial granules were investigated. Results showed that the substrate P/COD ratio had a significant effect on the elemental compositions of P-accumulating microbial granules. The P content in granules fell into a range of 1.9–9.3% by weight and increased with the increase of substrate P/COD ratio. Chemical precipitation-related P in granules accounted for less than 10% of the total P accumulation, i.e. biological storage was mainly responsible for the observed P accumulation in granules. These indicate that microbial granules cultivated had an excellent ability to uptake phosphorus from liquid solution. According to the elemental analyses, the empirical formulae of P-accumulating granules developed at different substrate P/COD ratios were generated. Significant difference in the elemental compositions between P-accumulating and non-P-accumulating granules was observed, indicating a shift in microbial association. A substantial accumulation of calcium and magnesium ions was found in the P-accumulating granules, and was closely related to the poly-phosphate accumulated in granules. The present results show potential application of microbial granules in enhanced biological phosphorus removal process.

Denitrification on poly- β-hydroxybutyrate in microbial granular sludge sequencing batch reactor

Microbial granules were successfully cultivated in an alternating aerobic–anaerobic sequencing batch reactor (SBR) for removing organic carbon and nitrogen. It was found that almost all input ammonium was converted to nitrite and nitrate in the aerobic phase, while the efficiency of denitrification was highly related to the availability of external carbon source in the anaerobic phase. Complete denitrification was achieved with sufficient supply of external carbon, while only partial denitrification was observed with no addition of external carbon. Results showed that in the absence of external carbon source, pre-accumulated poly- β-hydroxybutyric acid (PHB) in microbial granules could be utilized for cell maintenance and denitrification. With supply of external carbon but no addition of nitrate, PHB accumulation accounted for the main mechanism of the organic removal. Under balanced growth conditions (with organic carbon and nitrogen supply), external organic carbon was consumed simultaneously for denitrification, PHB storage as well as for cell functions. It was revealed that the potential role of PHB for denitrification by microbial granules was very limited, i.e. less than 28 mg nitrate-nitrogen l −1 was found to be denitrified with internally accumulated PHB. This study for the first time shows the limiting capacity of PHB as reducing power for denitrification by microbial granules.

Removal of micro-particles by microbial granules used for aerobic wastewater treatment

Microbial granules with a diameter from 0.4 mm to 3.0 mm have been produced by fast sedimentation and retention of microbial aggregates in sequencing batch airlift reactors used for model wastewater treatment. The wastewater was with or without addition of calcium salt. The granules were able not only to degrade organic matter but to remove nano- and micro-particles from wastewater due to microchannels and pores in the matrix of the granules. To detect the removal of 0.1 microm, 0.6 pm, 4.2 microm fluorescent microspheres, and cells of Escherichia coli, stained by permeable nucleic acid stain SYTO9, the granules were incubated with these particles. The rate of particle removal and their accumulation in the granules was measured by a Fluoview300 confocal laser scanning microscope (CLSM) (Olympus, Japan); a FACSCalibur flow cytometer (Becton Dickinson, CA, USA), and a fluorescence spectrometer LS-50B (Perkin-Elmer, UK). The release or removal of biological and non-biological particles was analyzed by a flow cytometer after DNA staining. Total number of the particles bigger than 0.1 microm in the reactors was approximately 4 x 10(7) per ml, and 23% of these particles were bacterial cells. The 0.1 microm and 4.2. microm microbeads were accumulated within 250 microm in the upper layer of the microbial granule but externally added cells of Escherichia coli penetrated to the depth of approximately 800 microm in the granules without calcium addition. Microbial granules contained also attached ciliates but accumulation of the particles in protozoan cells was smaller than in the granule matrix. Kinetics of particle sorption was revealed by flow cytometry and fluorescence spectrometry. Almost half of the stained cells of E. coli can be removed by the granules for one hour. The ability of the microbial granules to remove the particles can enhance their function in aerobic treatment of wastewater.

High-Rate Biodegradation of Phenol by Aerobically Grown Microbial Granules

This study demonstrates that aerobic granules can be developed to achieve high phenol loading rates in a sequencing batch reactor. The reactor was started at a loading rate of 1.5 kgphenolm-3d-1 with phenol-enriched activated sludge as inoculum. Granules first appeared on Day 9 after startup and quickly grew to become the dominant biomass in the reactor. The phenol loading was then adjusted stepwise to a final value of 2.5 kgphenolm-3d-1. At this high loading, phenol was completely degraded and high biomass concentration was maintained in the reactor. The biomass continued to possess a good settling ability, with a sludge volume index of 60.5 mLgSS-1 (SS stands for suspended solids). Granules remained stable, without significant deterioration in granule structure and physiology, even at the maximum phenol loading rate tested. The applied selection pressure enabled the micro-organisms to aggregate into granules, and the compact structure of the aerobic granules served both to retain biomass and protect the...

Rapid cultivation of stable aerobic phenol-degrading granules using acetate-fed granules as microbial seed

The aim of this study is to evaluate the utility of using aerobic acetate-fed microbial granules as a starting seed to rapidly develop stable aerobic phenol-degrading granules. Aerobic granules were first cultivated in four sequencing batch reactors with acetate as sole carbon source at a loading rate of 3.8 kg m −3 d −1. Phenol was then added to reactors R1, R2, R3 and R4 at loading rates of 0, 0.6, 1.2 and 2.4 kg m −3 d −1, respectively. The granules acclimated quickly to the phenol loading, and stabilized only 1 week after phenol was introduced. The granules exhibited good settling ability with good biomass retention and good metabolic activity, as evidenced by the low SVI values, stable biomass concentrations and good removal of acetate and phenol. No significant inhibitory effects from phenol toxicity were observed at the intermediate loadings of 0.6 and 1.2 kg phenol m −3 d −1, except for a slight lag in the ability of the granules to degrade phenol during the initial cycles. At the highest loading of 2.4 kg phenol m −3 d −1, a sharp buildup of phenol was observed in reactor R4 because the granules were initially unable to degrade phenol. However, this buildup quickly dissipated as the granules adapted rapidly to the high phenol concentrations. The compact structure of the acetate-fed granules likely protected the microorganisms against phenol toxicity and facilitated microbial acclimation towards faster phenol degradation rates. This is the first study to demonstrate the benefits of using aerobic granules cultivated on benign substrates as microbial seed to produce granules to degrade toxic substrates. This concept of using granules to produce different granules can be extended to granule-based applications involving other toxic chemicals and other types of high-strength industrial wastewaters, where rapid reactor start-up and system stability are key considerations.

반응성 염료폐수 처리를 위한 Comamonas sp. AEBL-85 분리 및 회분식 탈색

Azo계 염색 폐수 처리에 활용되었던 활성 슬러지의 미생물 군집체로부터 diazo계 반응성 염료인 Reactive Black 5를 유일 탄소원으로 성장할 수 있는 Comamonas sp. AEBL-85를 분리ㆍ동정하고, Comamonas sp. AEBL-85를 이용한 Reactive Slacks에 대한 회분식 탈색 특성을 평가하였다. 염료탈색 반응 효율 향상을 위해 보조 탄소원 및 질소원 첨가하고, pH, 온도 등의 분해조건이 탈색율에 미치는 영향을 분석한 결과, 3% (w/v)의 포도당, 0.5% (w/v)의 yeast extract를 첨가한 MSM에서 pH 6.0, 온도 35<TEX>$^{\circ}C$</TEX>의 조건에서 탈색효율이 가장 높았다. 초기농도 50 mg/l의 Reactive Black 5에 대하여 40시간의 회분식 탈색반응을 통해 약 95% 이상의 탈색율을 얻을 수 있었다. Comamonas sp. AEBL-85 was isolated from microbial granules in an activated sludge process of long-term operated for the treatment of reactive azo dye, and characterized its capability to decolorize Reactive Black 5. The effects of adding carbon source and nitrogen source on the extent of decol-orization were analyzed to develop an optimal medium. The optimum initial pH and temperature wire 6.0 and 35<TEX>$^{\circ}C$</TEX>, respectively. Reactive Black 5 of 50 mg/l was readily decolorized up to 95% within 40 hr by Comamonas sp. AEBL-85.

The effect of hydraulic retention time on the stability of aerobically grown microbial granules.

The aim of this study is to evaluate the effect of hydraulic retention time (HRT) on the development of aerobically grown microbial granules. Five column-shaped sequential aerobic sludge blanket reactors (SASBRs) were seeded with aerobically grown microbial granules and operated in a cyclic mode at different HRTs. At the shortest HRT of 1 h, the strong hydraulic pressure triggered biomass washout and led to reactor failure. At the longest HRT of 24 h, which represented the weakest hydraulic selection in this study, aerobic granules were gradually substituted by bioflocs because of the lower frequency of volumetric exchange. Within the optimum range of HRTs from 2 to 12 h, however, aerobic granules became stabilized in the presence of adequate hydraulic selection in the reactors, with good mixed liquor volatile suspended solids (MLVSS) retention, high volumetric chemical oxygen demand (COD) removal, low sludge volume index (SVI) values, good effluent quality, low sludge production rate, stronger and more compact structures, high cell hydrophobicity and high ratios of extracellular polysaccharides (PS) to extracellular proteins (PN). HRTs between 2 and 12 h provided the hydraulic selection pressures favourable for the formation and maintenance of stable aerobic granules with good settleability and activity. This is the first systematic study on the effect of HRT on heterotrophic aerobic granules. The results of the investigation are useful in understanding how aerobic granules can be applied for wastewater treatment.

A novel granular sludge sequencing batch reactor for removal of organic and nitrogen from wastewater

Microbial granules were developed at different substrate N/COD ratios in sequencing batch reactors (SBR). Results showed that heterotrophic, nitrifying, and denitrifying populations could peacefully co-exist in microbial granules, while increased substrate N/COD ratio led to significant shifts among three populations in granules. Enhanced activities of nitrifying and denitrifying populations were obtained in microbial granules developed at high substrate N/COD ratios, however, heterotrophic populations in granules tended to decrease with the increase of substrate N/COD ratio. It was found that dissolved oxygen (DO) concentration had a pronounced effect on the efficiency of denitrification by microbial granules, meanwhile the results also indicated that a certain mixing power would be provided to ensure mass transfer between liquid and granules during denitrification. It was demonstrated that complete organics and nitrogen removal can be achieved in single granule-based SBR with high efficiency and stable performance. This is the first study to show the capability of microbial granules in simultaneous removal of organic carbon and nitrogen from wastewater.

Presence of anaerobic bacteroides in aerobically grown microbial granules.

Microbial granules were grown in a column-type sequential aerobic sludge blanket reactor inoculated with activated sludge flocs taken from a wastewater treatment plant and containing a medium with glucose as the main carbon source. The reactor selected for granules that could settle rapidly by employing a short settling time of 2 min. Matured granules with diameters between 2 and 3 micro m were examined for anaerobic bacteria as their presence can signal the onset of diffusion limitation problems that can potentially diminish granule stability due to the bacterial production of fermentation gases and organic acids under anaerobic conditions. To detect the anaerobes in the granules, clones were constructed from 16S rRNA PCR amplicons. Two sequence types associated with a strict anaerobe Bacteroides spp. were identified from these clones. Fluorescence in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM) demonstrated that cells of Bacteroides spp. were concentrated at a depth of approximately 800 micro m below the surface of the granule. Cell enumeration using flow cytometry showed that the percentage of labeled cells of Bacteroides spp. compared to total bacterial cells in the granules was 0.56%. This is the first study to use a suite of culture-independent techniques to report the presence of a defined species of anaerobic bacteria in aerobically grown microbial granules.

Pilot-plant study on anaerobic treatment of a lipid- and protein-rich food industrial wastewater by a thermophilic multi-staged UASB reactor

An on-site pilot-scale experiment was conducted to investigate the performance of a multi-staged UASB (MS-UASB) reactor by feeding with a food processing wastewater containing high strength of lipid and protein. The reactor was operated at a thermophilic condition (55 degrees C) for a period of 600 days. The reactor finally achieved 50 kgCOD.m(-3) d(-1) with a soluble COD removal of 90% (based on the influent total COD versus the effluent filtered COD), while the overall COD removal (based on the effluent COD-total) as considerably unsatisfactory at around only 60-70%. The presence of high strength of lipid and protein along with high concentration of Mg and Ca ions in the raw wastewater caused a severe scum and/or insolubilized substance formation within the UASB sludge bed, resulting in hindering the contact efficiency between substrate and sludge. The replacement of active microbial granules in the sludge bed with the insolubilized protein and lipid brought about deterioration of sludge methanogenic activity.

Specific layers in aerobically grown microbial granules.

To determine the optimal size of aerobically grown granules for wastewater treatment by measuring specific layers within the granules. A variety of biological layers were detected by oligonucleotide probes, specific fluorochromes, and fluorescent microspheres. The channels in the granule matrix penetrated to depths of 900 microm. A layer of obligate anaerobic bacteria was detected at a depth of 800 microm below the granule surface. Dead cells were also observed in the granule interior. Aerobically grown granules contained layers of aerobic and anaerobic micro-organisms. The optimal diameter of the aerobic granule is less than 1600 microm. This is twice the distance from the granule surface to the anaerobic layer. This approach can be used to optimize the thickness of other microbial aggregates such as flocs, colonies and biofilms.

Effects of Storage on Viability and Efficacy of Granular Formulations of the Microbial Herbicides Alternaria alternata and Trematophoma lignicola

Alternaria alternata Pesta (gluten matrix) granules stored at 12% relative humidity were still viable and infective, giving total control of Amaranthus retroflexus after 24 months storage. Viability of the propagule in the granule was an important factor of the evaluation test and not just the viability of the granule. The concentration of inoculum in the granule was important, with 10 5 to 10 6 conidia g -1 being the ideal, since lower concentrations may not be able to out-compete the soil micro-flora. Even with soil application, the effect of local humidity was still important. Trematophoma lignicola was also formulated as microbial herbicide granules but the conidia were severely damaged in the production process.

Dechlorination of spiked PCBs in lake sediment by anaerobic microbial granules

An anaerobic microbial consortium developed in our laboratory showed dechlorination of spiked polychlorinated biphenyls (PCBs), such as Aroclor 1254 and a defined congener 2,3,4,5,6-pentachlorobiphenyl (CB) in the presence and the absence of lake sediment. Glucose and methanol were used as carbon and energy sources. Highly chlorinated congeners (containing >5 chlorines) such as penta-, hexa- and heptachlorobiphenyls were preferentially dechlorinated both in the presence and the absence of sediment with a simultaneous change in the distribution of relative amounts of tri- and tetrachlorobiphenyl congeners. The dechlorination pattern and rates observed in both sediment and sediment-free conditions were similar. In 24weeks, almost 70% of the PCBs were dechlorinated without accumulation of any specific PCB congeners. Monochlorobiphenyls were dechlorinated at a higher rate than di- and tetrachlorobiphenyls.

Anaerobic dechlorination of perchloroethylene (PCE) in soil by a dechlorinating microbial consortium

An anaerobic dechlorinating microbial granular consortium was developed for biotreatment of soil contaminated with perchloroethylene (PCE) and trichloroethylene (TCE). Microcosm tests were conducted at ambient temperature (22°C) to examine the performance of PCE dechlorination by this consortium. TCE, dichloroethylene (DCE), and vinyl chloride (VC) were transiently observed as intermediates. Ethylene was the end product of PCE dechlorination. After four months' incubation with the consortium, ethane was observed as a product from reduction of ethylene in microcosms spiked with a low level of PCE (10 mg/kg soil) but not in those with a high PCE level (200 mg/kg soil). At the end of five months, more than 90% of the PCE added to the soil was accounted for, on a molar basis, as ethylene and ethane. The dechlorination was predominately supported by soil organic matter. The dechlorination performance of the consortium was not inhibited whether the soil was spiked with 10 or 200 mg PCE/kg dry soil. The inoculum level influenced the initial dechlorination process, but there was no significant difference in the extent of dechlorination after three months at a low or high level of inoculum (3 or 60 mg VSS/kg soil). Partial and slow dechlorination was also observed in controls without added inoculum due to activity of indigenous soil organisms. In the controls, TCE, cis-1,2-DCE, and VC ware accumulated as intermediates. No dechlorination occurred in soil that was autoclaved and did not receive microbial granules. Methanogenesis was inhibited in the presence of the high initial PCE level (200 mg/kg soil) but not at the low level (10 mg/kg soil).

Strength characterisation of microbial granules

The strength of microbial granules is still poorly understood. The granule strength is defined as the resistance to attrition and/or breaking by a mechanical force or the liquid shear stress. The strength of various types of microbial granules were studied. The objective was to develop an in vitro strength characterisation test and to be able to predict granule behaviour in full scale systems. Abrasion experiments were conducted in stirred tanks and bubble columns, while the influence of viscosity and medium composition, shear rate and particle size were studied. The medium viscosity did not influence the relation between shear rate and abrasion. Measurements in demineralized water or media containing complexing agents resulted in lower granule strengths presumably the result of the removal of calcium from the granule matrix. In bubble columns attrition is the predominant abrasion process. In full scale reactors, e.g. UASB, attrition is apparently insignificant. In stirred tanks also granule breakage was observed. The abrasion rate is not directly related to the stirrer tip speed. Larger methanogenic granules showed more abrasion compared to small ones. In contrast larger nitrifying granules were stronger than small ones. A standardized strength characterization test was developed to be able to compare strengths of different microbial granules. The strength of slow growing systems appeared to be higher as compared to fast growing systems. From an ecological point of view, cells growing slowly in continuous operated systems gain advantage from attachment and shear resistance or strength.

Reductive dechlorination of PCB‐contaminated raisin river sediments by anaerobic microbial granules

A polychlorinated biphenyl (PCB)-dechlorinating anaerobic microbial consortium, developed in a granular form, demonstrated extensive dechlorination of PCBs present in Raisin River sediments at room (20° to 22°C) and at a relatively low (12°C) temperature. Highly chlorinated PCB congeners were dechlorinated and less chlorinated compounds were produced. The homolog comparison showed that tri-, tetra-, penta-, hexa-, and heptachlorobiphenyl compounds decreased significantly, and mono- and dichlorobiphenyl compounds increased. After 32 weeks of incubation at 12°C, the predominant less chlorinated products included 2-, 4-, 2-2/26-, 24-, 2-4-, 24-2-, 26-2-, and 26-4-CB. Among these, 24- and 24-2-CB did not accumulate at room temperature, suggesting a further dechlorination of these congeners. Predominantly meta dechlorination (i.e., pattern M) was catalyzed by the microbial consortium in the granules. Dechlorination in the control studies without granules was not extensive. This study is the first demonstration of enhanced reductive dechlorination of sediment PCBs by an exogenous anaerobic microbial consortium. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 182–190, 1997.

Reductive dechlorination of PCB-contaminated raisin river sediments by anaerobic microbial granules

A polychlorinated biphenyl (PCB)-dechlorinating anaerobic microbial consortium, developed in a granular form, demonstrated extensive dechlorination of PCBs present in Raisin River sediments at room (20 degrees to 22 degrees C) and at a relatively low (12 degrees C) temperature. Highly chlorinated PCB congeners were dechlorinated and less chlorinated compounds were produced. The homolog comparison showed that tri-, tetra-, penta-, hexa-, and heptachlorobiphenyl compounds decreased significantly, and mono- and dichlorobiphenyl compounds increased. After 32 weeks of incubation at 12 degrees C, the predominant less chlorinated products included 2-, 4-, 2-2/26-, 24-, 2-4-, 24-2-, 26-2-, and 26-4-CB. Among these, 24- and 24-2-CB did not accumulate at room temperature, suggesting a further dechlorination of these congeners. Predominantly meta dechlorination (i.e., pattern M) was catalyzed by the microbial consortium in the granules. Dechlorination in the control studies without granules was not extensive. This study is the first demonstration of enhanced reductive dechlorination of sediment PCBs by an exogenous anaerobic microbial consortium.