MAPKs trigger antiviral immunity by directly phosphorylating a rhabdovirus nucleoprotein in plants and insect vectors.

Signaling by the evolutionarily conserved mitogen-activated protein kinase or extracellular signal-regulated kinase (MAPK/ERK) plays critical roles in converting extracellular stimuli into immune responses. However, whether MAPK/ERK signaling induces virus immunity by directly phosphorylating viral effectors remains largely unknown. Barley yellow striate mosaic virus (BYSMV) is an economically important plant cytorhabdovirus that is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. Here, we found that the barley (Hordeum vulgare) MAPK MPK3 (HvMPK3) and the planthopper ERK (LsERK) proteins interact with the BYSMV nucleoprotein (N) and directly phosphorylate N protein primarily on serine 290. The overexpression of HvMPK3 inhibited BYSMV infection, whereas barley plants treated with the MAPK pathway inhibitor U0126 displayed greater susceptibility to BYSMV. Moreover, knockdown of LsERK promoted virus infection in SBPHs. A phosphomimetic mutant of the N Ser290 (S290D) completely abolished virus infection because of impaired self-interaction of BYSMV N and formation of unstable N-RNA complexes. Altogether, our results demonstrate that the conserved MAPK and ERK directly phosphorylate the viral nucleoprotein to trigger immunity against cross-kingdom infection of BYSMV in host plants and its insect vectors.

A rhabdovirus accessory protein inhibits jasmonic acid signaling in plants to attract insect vectors.

Plant rhabdoviruses heavily rely on insect vectors for transmission between sessile plants. However, little is known about the underlying mechanisms of insect attraction and transmission of plant rhabdoviruses. In this study, we used an arthropod-borne cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), to demonstrate the molecular mechanisms of a rhabdovirus accessory protein in improving plant attractiveness to insect vectors. Here, we found that BYSMV-infected barley (Hordeum vulgare L.) plants attracted more insect vectors than mock-treated plants. Interestingly, overexpression of BYSMV P6, an accessory protein, in transgenic wheat (Triticum aestivum L.) plants substantially increased host attractiveness to insect vectors through inhibiting the jasmonic acid (JA) signaling pathway. The BYSMV P6 protein interacted with the constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5) of barley plants in vivo and in vitro, and negatively affected CSN5-mediated deRUBylation of cullin1 (CUL1). Consequently, the defective CUL1-based Skp1/Cullin1/F-box ubiquitin E3 ligases could not mediate degradation of jasmonate ZIM-domain proteins, resulting in compromised JA signaling and increased insect attraction. Overexpression of BYSMV P6 also inhibited JA signaling in transgenic Arabidopsis (Arabidopsis thaliana) plants to attract insects. Our results provide insight into how a plant cytorhabdovirus subverts plant JA signaling to attract insect vectors.

Casein Kinase 1 Regulates Cytorhabdovirus Replication and Transcription by Phosphorylating a Phosphoprotein Serine-Rich Motif.

Casein kinase 1 (CK1) family members are conserved Ser/Thr protein kinases that regulate important developmental processes in all eukaryotic organisms. However, the functions of CK1 in plant immunity remain largely unknown. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, infects cereal crops and is obligately transmitted by the small brown planthopper (SBPH; Laodelphax striatellus). The BYSMV phosphoprotein (P) exists as two forms with different mobilities corresponding to 42 kD (P42) and 44 kD (P44) in SDS-PAGE gels. Mass spectrometric analyses revealed a highly phosphorylated serine-rich (SR) motif at the C-terminal intrinsically disordered region of the P protein. The Ala-substitution mutant (PS5A) in the SR motif stimulated virus replication, whereas the phosphorylation-mimic mutant (PS5D) facilitated virus transcription. Furthermore, PS5A and PS5D associated preferentially with nucleocapsid protein-RNA templates and the large polymerase protein to provide optimal replication and transcription complexes, respectively. Biochemistry assays demonstrated that plant and insect CK1 protein kinases could phosphorylate the SR motif and induce conformational changes from P42 to P44. Moreover, overexpression of CK1 or a dominant-negative mutant impaired the balance between P42 and P44, thereby compromising virus infections. Our results demonstrate that BYSMV recruits the conserved CK1 kinases to achieve its cross-kingdom infection in host plants and insect vectors.

Ynamide-Mediated Thioamide and Primary Thioamide Syntheses.

Environmentally friendly ynamide-mediated thioamidation of monothiocarboxylic acids with amines or ammonium hydroxide for the syntheses of thioamides and primary thioamides is described. Simple and mild reaction conditions enable the reaction to tolerate a wide variety of functional groups such as hydroxyl group, ester, tertiary amine, ketone, and amide moieties. Readily available NaSH served as the sulfur source, avoiding the use of toxic, expensive, and malodorous organic sulfur reagents and making this strategy environmentally friendly and practical. Importantly, the stereochemical integrity of α-chiral monothiocarboxylic acids was maintained during the activation step and subsequent aminolysis process, thus offering a racemization-free strategy for peptide C-terminal modification. Furthermore, a number of thioamide-modified drugs were prepared in good yields by using this protocol and the synthesized primary thioamides were transformed into backbone thiazolyl modified peptides.

Molecular Characteristics of Subgenomic RNAs and the Cap-Dependent Translational Advantage Relative to Corresponding Genomic RNAs of Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) causes severe viral diseases on many economically important plants of Solanaceae. During the infection process of TSWV, a series of 3'-truncated subgenomic RNAs (sgRNAs) relative to corresponding genomic RNAs were synthesized, which were responsible for the expression of some viral proteins. However, corresponding genomic RNAs (gRNAs) seem to possess the basic elements for expression of these viral proteins. In this study, molecular characteristics of sgRNAs superior to genomic RNAs in viral protein expression were identified. The 3' ends of sgRNAs do not cover the entire intergenic region (IGR) of TSWV genomic RNAs and contain the remarkable A-rich characteristics. In addition, the 3' terminal nucleotides of sgRNAs are conserved among different TSWV isolates. Based on the eIF4E recruitment assay and subsequent northern blot, it is suggested that the TSWV sgRNA, but not gRNA, is capped in vivo; this is why sgRNA is competent for protein expression relative to gRNA. In addition, the 5' and 3' untranslated region (UTR) of sgRNA-Ns can synergistically enhance cap-dependent translation. This study further enriched the understanding of sgRNAs of ambisense RNA viruses.

Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies.

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid ß-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.

A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43-55) and the remaining region (aa 56-295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

Inhibitory effect of high phenol concentration in treating coal gasification wastewater in anaerobic biofilter.

In this paper, the inhibition of methanogens by phenol in coal gasification wastewater (CGW) was investigated by both anaerobic toxicity tests and a lab-scale anaerobic biofilter reactor (AF). The anaerobic toxicity tests indicated that keeping the phenol concentration in the influent under 280mg/L could maintain the methanogenic activity. In the AF treating CGW, the result showed that adding glucose solution as co-substrate could be beneficial for the quick start-up of the reactor. The effluent chemical oxygen demand (COD) and total phenol reached 1200 and 100mg/L, respectively, and the methane production rate was 175mLCH4/gCOD/day. However, if the concentration of phenol was increased, the inhibition of anaerobic micro-organisms was irreversible. The threshold of total phenol for AF operation was 200-250mg/L. The extracellular polymeric substances (EPS) and particle size distribution of anaerobic granular sludge in the different stages were also examined, and the results indicated that the influence of toxicity in the system was more serious than its effect on flocculation of EPS. Moreover, the proportion of small size anaerobic granular sludge gradually increased from 10.2% to 34.6%. The results of high through-put sequencing indicated that the abundance of the Chloroflexi and Planctomycetes was inhibited by the toxicity of the CGW, and some shifts in the microbial community were observed at different stages.

Identification of three new isolates of Tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses.

BACKGROUND: Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide. METHODS: RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. RESULTS: Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5' regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees. CONCLUSIONS: All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative-sense RNA viruses.

An advanced anaerobic biofilter with effluent recirculation for phenol removal and methane production in treatment of coal gasification wastewater.

An advanced anaerobic biofilter (AF) was introduced for the treatment of coal gasification wastewater (CGW), and effluent recirculation was adopted to enhance phenol removal and methane production. The results indicated that AF was reliable in treating diluted CGW, while its efficiency and stability were seriously reduced when directly treating raw CGW. However, its performance could be greatly enhanced by effluent recirculation. Under optimal effluent recirculation of 0.5 to the influent, concentrations of chemical oxygen demand (COD) and total phenol in the effluent could reach as low as 234.0 and 14.2mg/L, respectively. Also, the rate of methane production reached 169.0mLCH4/L/day. Though CGW seemed to restrain the growth of anaerobic microorganisms, especially methanogens, the inhibition was temporary and reversible, and anaerobic bacteria presented strong tolerance. The activities of methanogens cultivated in CGW could quickly recover on feeding with glucose wastewater (GW). However, the adaptability of anaerobic bacteria to the CGW was very poor and the activity of methanogens could not be improved by long-term domestication. By analysis using the Haldane model, it was further confirmed that high effluent recirculation could result in high activity for hydrolytic bacteria and substrate affinity for toxic matters, but only suitable effluent recirculation could result in high methanogenic activity.

Characteristics of pellets with immobilized activated sludge and its performance in increasing nitrification in sequencing batch reactors at low temperatures.

Immobilized pellets obtained by means of entrapping activated sludge in waterborne polyurethane were successfully adapted in ammonium (NH4(+)-N) synthetic wastewater. Its physicochemical characteristics were determined using scanning electron microscope, pyrosequencing, and microelectrodes, and its influence on the nitrification process in sequencing batch reactors (SBRs) at low temperatures was evaluated. A large number of rod-shaped bacteria were observed on the surface of the immobilized pellet, in which Rudaea spp. (Xanthomonadaceae family) was an important bacterial component (23.44% of the total bacteria). The oxygen uptake rate of immobilized pellets reached 240.83±15.59 mgO2/(L·hr), and the oxygen was primarily consumed by the bacteria on the pellet surfaces (0-600 µm). The dosing of the pellets (30 mL/L) into an SBR significantly improved the nitrification efficiency at low temperatures of 7-11°C, achieving an average NH4(+)-N removal of 84.09%, which is higher than the removal of 67.46% observed for the control group.

Phosphate removal from water by a novel zeolite/lanthanum hydroxide hybrid material prepared from coal fly ash.

This study was undertaken to investigate the effectiveness of the hybrid adsorbent, which was synthesized from coal fly ash and was composed of lanthanum hydroxide and zeolite (La-ZFA), for phosphate removal from water. Long-term repeated adsorption tests for 30 days showed that the maximum removal capacity of the material reached 66.09 mg P/g. The fractionation of adsorbed phosphorus indicated that phosphate immobilized by La-ZFA was quite irreversible and was dominated by HCl-P fraction. It was suggested that the immobilization of phosphate was mainly attributed to lanthanum hydroxide and was slightly influenced by coexistence of other anions (Cl(-), NO3(-), SO4(2-), and HCO3(-)). At a La/P molar ratio between 1.5:1 and 2.0:1, a nearly complete removal (above 98%) of phosphate could be achieved. La-ZFA also exhibited great performance for removing phosphate from lake water (97.29%) as well as the effluent from wastewater treatment plant (97.86%), respectively. In addition, based on the results of the present study, it was believed that La-ZFA could be a potential material for phosphate removal in practical application.

Effect of substrates and free ammonia on kinetic characteristics of nitritation and nitratation by entrapped nitrifiers.

In the present study, nitrifying bacteria entrapped in waterborne polyurethane gel was used to investigate the kinetic characteristics of nitritation and nitratation in relation to achieve shortcut nitrification. The nitrite accumulation rate was over 80% during the acclimation period. The following kinetic parameters were experimentally obtained: maximum nitrification rate (v(max)), half-saturation coefficient (K(s) and K(o)), and inhibition coefficient (K(IH)). The bacterial populations were also determined by fluorescence in situ hybridization. 73.5% proportion of ammonia oxidizing bacteria (AOB) resulted in a significantly higher ammonia oxidizing rate than nitrite oxidizing rate, which is in agreement with higher V(max) of nitritation (608.5 mgNl(-1)-pellet h(-1)) over nitratation (66.3 mgN l(-1)-pellet h(-1)).

Screening novel candidates of ZL003-based organic dyes for dye-sensitized solar cells by modifying auxiliary electron acceptors: A theoretical study.

In this work, a series of ZL003-based free-metal sensitizers with the donor-acceptor-π- conjugated spacer-acceptor (D-A-π-A) structure were designed by modifying auxiliary electron acceptors for the potential application in dye-sensitized solar cells. The energy levels of frontier molecular orbitals, absorption spectra, electronic transition, and photovoltaic parameters for all studied dyes were systematically evaluated using density functional theory (DFT)/time-dependent DFT calculations. Results illustrated that thienopyrazine (TPZ), selenadiazolopyridine (SDP), and thiadiazolopyridine (TDP) are excellent electron acceptors, and dye sensitizers functionalized by these acceptors have smaller HOMO-LUMO gaps, obviously red-shifted absorption bands and stronger light harvesting. The present study revealed that the photoelectric conversion efficiency (PCE) of ZL003 is around 13.42 % with a JSC of 20.21 mA·cm-2, VOC of 966 mV and FF of 0.688 under the AM 1.5G sun exposure, in good agreement with its experimental value (PCE = 13.6 ± 0.2 %, JSC = 20.73 ± 0.20 mA·cm-2, VOC = 956 ± 5 mV, and FF = 0.685 ± 0.005.). With the same procedure, the PCE values for M4, M6, and M7 were estimated to be as high as 19.93 %, 15.38 %, and 15.80 % respectively. Hence, these three dyes are expected to be highly efficient organic sensitizers applied in practical DSSCs.

Occurrence and distribution of microcystins in Lake Taihu, China.

The occurrence and distribution of microcystins were investigated in Lake Taihu, the third largest lake in China. An extensive survey, larger and broader in scale than previous studies, was conducted in summer 2010. The highest microcystin concentration was found at southern part of Taihu, which was newly included in this survey. In northern coastal areas, total cellular concentrations of 20 to 44 µg/L were observed. In northern offshore waters, levels were up to 4.8 µg/L. Microcystin occurrence was highly correlated with chemical oxygen demand, turbidity, and chlorophyll-a. Extracellular/total cellular microcystin (E/T) ratios were calculated and compared to other water quality parameters. A higher correlation was found using E/T ratios than original microcystin values. These results show that algal blooms are having a severe impact on Lake Taihu, and further and extensive monitoring and research are required to suppress blooms effectively.

Nitrification characteristics of nitrobacteria immobilized in waterborne polyurethane in wastewater of corn-based ethanol fuel production.

A technology to achieve stable and high ammonia nitrogen removal rates for corn distillery wastewater (ethanol fuel production) treatment has been designed. The characteristics of nitrifying bacteria entrapped in a waterborne polyurethane (WPU) gel carrier were evaluated after acclimation. In the acclimation period, nitrification rates of WPU-immobilized nitrobacteria were monitored and polymerase chain reaction (PCR) was also carried out to investigate the change in ammonium-oxidizing bacteria. The results showed that the pellet nitrification rates increased from 21 to 228 mg-N/(L-pellet x hr) and the quantity of the ammonia oxidation bacteria increased substantially during the acclimation. A continuous ammonia removal experiment with the anaerobic pond effluent of a distillery wastewater system was conducted with immobilized nitrifying bacteria for 30 days using an 80 L airlift reactor with pellets at a fill ratio of 15% (V/V). Under the conditions of 75 mg/L influent ammonia, hydraulic retention time (HRT) of 3.7-5.6 hr, and dissolved oxygen (DO) of 4 mg/L, the effluent ammonia concentration was lower than 10 mg/L and the ammonia removal efficiency was 90%. While the highest ammonia removal rate, 162 mg-N/(L-pellet x hr), was observed when the HRT was 1.3 hr.

Nitrification performance of nitrifying bacteria immobilized in waterborne polyurethane at low ammonia nitrogen concentrations.

Suspended and waterborne polyurethane immobilized nitrifying bacteria have been adopted for evaluating the effects of environmental changes, such as temperature, dissolved oxygen (DO) concentration and pH, on nitrification characteristics under conditions of low ammonia concentrations. The results showed that nitrification was prone to complete with increasing pH, DO and temperature. Sensitivity analysis demonstrated the effects of temperature and pH on nitrification feature of suspended bacteria were slightly greater than those of immobilized nitrifying bacteria. Immobilized cells could achieve complete nitrification at low ammonia concentrations when DO was sufficient. Continuous experiments were carried out to discuss the removal of ammonia nitrogen from synthetic micropollute source water with the ammonia concentration of about 1 mg/L using immobilized nitrifying bacteria pellets in an up-flow inner circulation reactor under different hydraulic retention times (HRT). The continuous removal rate remains above 80% even under HRT 30 min. The results verified that the waterborne polyurethane immobilized nitrifying bacteria pellets had great potential applications for micro-pollution source water treatment.

Autotrophic denitrification for nitrate and nitrite removal using sulfur-limestone.

Sulfur-limestone was used in the autotrophic denitrification process to remove the nitrate and nitrite in a lab scale upflow biofilter. Synthetic water with four levels of nitrate and nitrite concentrations of 10, 40, 70 and 100 mg N/L was tested. When treating the low concentration of nitrate- or nitrite-contaminated water (10, 40 mg N/L), a high removal rate of about 90% was achieved at the hydraulic retention time (HRT) of 3 hr and temperature of 20-25 degrees C. At the same HRT, 50% of the nitrate or nitrite could be removed even at the low temperature of 5-10 degrees C. For the higher concentration nitrate and nitrite (70, 100 mg N/L), longer HRT was required. The batch test indicated that influent concentration, HRT and temperature are important factors affecting the denitrification efficiency. Molecular analysis implied that nitrate and nitrite were denitrified into nitrogen by the same microorganisms. The sequential two-step-reactions from nitrate to nitrite and from nitrite to the next-step product might have taken place in the same cell during the autotrophic denitrification process.

Analysis of differences in chemical properties of reconstructed soil under different proportions of topsoil substitute materials.

The purpose of this study was to evaluate the soil substitutes in land reclamation following mining activities. This study revealed different reconstructed soil materials have different effects on the chemical properties of reconstructed soil and explored the appropriate proportions of reconstructed soil materials. Using topsoil, coal gangue, fly ash, and rock and soil stripping material from the Shengli Mining Area of Inner Mongolia in China as raw materials, potting experiments were carried out according to different proportions. The variance analysis method was used to analyze the difference in the soil pH values, soil organic matter (SOM) content, soil total nitrogen (STN) content, soil available phosphorus (SAP) content and soil available potassium (SAK) content of the reconstructed soil, and the reasons for the difference were discussed. The results showed that in the treatment group where the topsoil substitute material was coal gangue, when the coal gangue content was 30%, 40%, and 50%, the pH values of reconstructed soil were lower than that of the control scheme. When the coal gangue content exceeded 10%, the soil nutrient status was obviously improved. In the treatment group where the topsoil substitute material was fly ash, the chemical properties of reconstructed soil tended to deteriorate. In the treatment group where the topsoil substitute material was rock and soil stripping material, when the content of rock and soil stripping material exceeded 20%, the chemical properties of reconstructed soil were mostly better than those of the control scheme. The study of the different proportions of coal gangue, fly ash, and rock and soil stripping material as topsoil substitute materials provide support for reclamation work in mining areas where topsoil is scarce, as well as a reference for ecological restoration projects in grassland open-pit mining areas around the world.

Anaerobic biological treatment of alginate production wastewaters in a pilot-scale expended granular sludge bed reactor under moderate to low temperatures.

Psychrophilic anaerobic digestion recently has been demonstrated as a cost-effective option for the treatment of a range of wastewater categories. In this study, the treatment of alginate production wastewaters was carried out in a pilot-scale expended granular sludge bed (EGSB) reactor. After a 40-day startup with two inocula, a 163-day experiment was run, from moderate to low temperatures, to treat seaweed-based-production wastewater. The results showed that inoculating with the active granular sludge instead of flocculent biomass can remarkably speed up the startup, and, at applied organic loading rates of 1.5 to 3.0 kg chemical oxygen demand (COD)/m3 x d, COD removal efficiencies of 55.4 to 72.6% were achieved. The volatile suspended solids ratio decreased slowly with operation time, as a result of the extremely slow growth rates of microorganisms and the accumulation of inorganic substances. Morphological examination and particle-size distribution of the granules revealed their tendency to disintegrate. Inorganic precipitates, microorganism shift, and substrate limitations may have contributed to it.