Cultivation and In Situ Detection of a Thermophilic Bacterium Capable of Oxidizing Propionate in Syntrophic Association with Hydrogenotrophic Methanogens in a Thermophilic Methanogenic Granular Sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55°C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor

High-rate biological conversion of sulfide and nitrate in synthetic wastewater to, respectively, elemental sulfur (S⁰) and nitrogen-containing gas (such as N₂) was achieved in an expanded granular sludge bed (EGSB) reactor. A novel strategy was adopted to first cultivate mature granules using anaerobic sludge as seed sludge in sulfate-laden medium. The cultivated granules were then incubated in sulfide-laden medium to acclimate autotrophic denitrifiers. The incubated granules converted sulfide, nitrate, and acetate simultaneously in the same EGSB reactor to S⁰, N-containing gases and CO₂ at loading rates of 3.0 kg S m⁻³ d⁻¹, 1.45 kg N m⁻³ d⁻¹, and 2.77 kg Ac m⁻¹ d⁻¹, respectively, and was not inhibited by sulfide concentrations up to 800 mg l⁻¹. Effects of the C/N ratio on granule performance were identified. The granules cultivated in the sulfide-laden medium have Pseudomonas spp. and Azoarcus sp. presenting the heterotrophs and autotrophs that co-work in the high-rate EGSB-SDD (simultaneous desulfurization and denitrification) reactor.     

Selectively inducing the synthesis of a key structural exopolysaccharide in aerobic granules by enriching for <Emphasis Type="BoldItalic">Candidatus</Emphasis> “<Emphasis Type="BoldItalic">Competibacter phosphatis</Emphasis>”

A gel-forming exopolysaccharide was previously shown to play an important structural role in aerobic granules treating nutrient-rich industrial wastewater. To identify whether this exopolysaccharide performs a similar role in other granular biomass and if conditions favouring its production can be more precisely elucidated, extracellular polymeric substances (EPS) were extracted from granules grown under four different operating conditions. ¹H nuclear magnetic resonance (NMR) spectroscopy of their EPS indicated that the gel-forming exopolysaccharide was expressed in two granular sludges both enriched in Candidatus “Competibacter phosphatis”. In contrast, it was not expressed in granules performing denitrification with methanol as a carbon source and nitrate as the electron acceptor or granules enriched in Candidatus “Accumulibacter phosphatis” performing enhanced biological phosphorus removal from synthetic wastewater. In one of the first two sludges, the exopolysaccharide contained in the seeding granular sludge continued to be a major component of the granule EPS while Competibacter was being enriched. In the second sludge, a floccular sludge not containing the gel-forming exopolysaccharide initially was also enriched for Competibacter. In this sludge, an increase in particle size was detected coinciding with a yield increase of EPS. NMR spectroscopy confirmed its yield increase to be attributable to the production of this structural gel-forming exopolysaccharide. The results show that (1) the particular gel-forming exopolysaccharide previously identified is not necessarily a key structural exopolysaccharide for all granule types, and (2) synthesis of this exopolysaccharide is induced under conditions favouring the selective enrichment of Competibacter. This indicates that Competibacter may be involved in its production.     

Biosorption and biodegradation of pentachlorophenol (PCP) in an upflow anaerobic sludge blanket (UASB) reactor

In order to understand the fate of PCP in upflow anaerobic sludge blanket reactor (UASB) more completely, the sorption and biodegradation of pentachlorophenol (PCP) by anaerobic sludge granules were investigated. The anaerobic granular sludge degrading PCP was formed in UASB reactor, which was seeded with anaerobic sludge acclimated by chlorophenols. At the hydraulic retention time (HRT) of 20–22 h, and PCP loading rate of 200–220 mg l⁻¹ d⁻¹, UASB reactor exhibited good performance in treating wastewater which containing 170–180 mg l⁻¹ PCP and the PCP removal rate of 99.5% was achieved. Sequential appearance of tetra-, tri-, di-, and mono-chlorophenol was observed in the reactor effluent after 20 mg l⁻¹ PCP introduction. Sorption and desorption of PCP on the anaerobic sludge granules were all fitted to the Freundlich isotherm equation. Sorption of PCP was partly irreversible. The Freundlich equation could describe the behavior of PCP amount sorbed by granular sludge in anaerobic reactor reasonably well. The results demonstrated that the main mechanism leading to removal of PCP on anaerobic granular sludge was biodegradation, not sorption or volatization.     

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     

Effects of physico-chemical factors on the viscosity evolution of anaerobic granular sludge

The rheological properties of anaerobic granular sludge samples from four full-scale and one lab-scale anaerobic bioreactors were characterized by determining their “limit viscosity” values. These values were deducted from the evolution of the apparent viscosity of granular sludge samples (20 mL) at steady shear rate (200 s⁻¹) recorded using rotation tests with a wings type measurement cell stirrer Anton-Parr reference: ST24-1D/2V-Q0. The limit viscosity values depended on the applied shear rate, indicating a non-Newtonian behavior of the anaerobic granular sludge types investigated. The effect of variations of physico-chemical parameters such as pH (involving surface charge change), size, surface roughness and TSS content on the evolution of the limit viscosity of an anaerobic granular sludge suspension was investigated. This showed the importance of both quantitative (number of particles in a given volume) as well as qualitative (surface charge or shape) granule-granule interactions on this rheological parameter. Moreover, the origin of the granular sludge strongly influenced the limit viscosity value according with different granules characteristics. This work confirms the ability of the rheological parameter “limit viscosity” as an overall parameter to describe the physico-chemical characteristics (TSS, granulometry, origin, and charge) of anaerobic granular sludge and showed this holds for both sieved (500 μm) and unsieved sludges.     

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Isolation and characterization of a carbon monoxide utilizing strain of the acetogen Peptostreptococcus productus

From sludge obtained from the sewage digester plant in Marburg-Cappel a strictly anaerobic bacterium was enriched and isolated with carbon monoxide as the sole energy source. Based on morphological and physiological characteristics the isolate was identified as a strain of Peptostreptococcus productus, which was called strain Marburg. The organism was able to grow on CO (50% at 200 kPa) as the sole energy source at a doubling time of 3 h and converted this substrate to acetate and CO₂. The type strain of P. productus was not able to grow at the expense of CO. Electron microscopic investigations of strain Marburg cells revealed a cell wall which was different from that of other Gram-positive prokaryotes. DNA:DNA hybridization studies of the DNA isolated from strain Marburg and the type strain as well as some morphological and physiological properties of both strains confirmed the low degree or relatedness between the two strains.     

The influence of organic load on granular sludge development in an acetate-fed system

Summary The development of granular sludge in laboratory-scale upflow anaerobic sludge-blanket reactors was studied. Acetate was supplied as sole carbon source in order to select the acetotrophs Methanosarcina and Methanothrix. These microorganisms are dominant in methanogenic ecosystems and their ratio seems to control the speed of granulation. Changing the ratio of the above species was followed on the basis of their different F ₄₂₀-coenzyme content. Five reactors were operated at the same hydraulic retention time but at different feed substrate concentrations. We found that granulation takes place only in acetate-fed systems but this process was slower and the resultant granules looser and less stable than those developed on sugar-starch substrate. In the range of feed acetate levels examined (0.5–0.3 g/1) higher concentrations of feed caused faster granulation of the sludge bed and, presumably, of the microbial population, and resulted in larger granules containing sludge that settled more readily. We found no evidence for selection pressure at substrate concentrations below 0.5 g/1 acetate in the reactor. Offprint requests to: J. Holló     

Long-term effects of multi-walled carbon nanotubes on the performance and microbial community structures of an anaerobic granular sludge system

Multi-walled carbon nanotubes (MWCNTs) released into the sewage may cause negative and/or positive effects on the treatment system. The objective of this study was to explore over 110 days’ effect of MWCNTs on the performance of anaerobic granular sludge and microbial community structures in an upflow anaerobic sludge blanket (UASB) reactor. The results showed that MWCNTs had no significant effect on the removal of chemical oxidation demand (COD) and ammonia in UASB reactor, but the total phosphorus (TP) removal efficiency increased by 29.34%. The biogas production of the reactor did not change. The anaerobic granular sludge tended to excrete more EPS to resist the effects of MWCNTs during the long-term impact. Illumina MiSeq sequencing of 16S rRNA gene revealed that MWCNTs did not affect the microbial diversity, but altered the composition and structure of microbial community in the reactor. In this process, Saccharibacteria replaced Proteobacteria as the highest abundant bacterial phylum. MWCNTs promoted the differentiation of methanogen structure, resulting in increase of Methanomassiliicoccus, Methanoculleus, and the uncultured WCHA1–57. These results indicated that MWCNTs impacted the performance of UASB reactor and the structures of the microbial community in anaerobic granular sludge.

     

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.     

Isolation and Characterization of Pseudoxanthomonas sp. Strain YP1 Capable of Denitrifying Phosphorus Removal (DPR)

A bacterial strain (designated as YP1) was isolated from an aerobic granular sequence batch reactor (SBR) performing simultaneous nitrogen and phosphorus removal. Based on the morphological, biochemical characteristics, and phylogenetic analysis of 16S rRNA gene sequence, YP1 was identified as Pseudoxanthomonas sp. strain. Strain YP1 was confirmed to have the ability to conduct denitrifying phosphorus removal (DPR). The optimal conditions for YP1 were pH 8.0, phosphorus (PO₄^3?-P) concentration of 8.0 mg/L, sodium citrate as carbon source, and nitrate nitrogen (NO₃^?-N) concentration of 30 mg/L. The functional genes including ppk and ppx, narG and narA, nirS and nirK were amplified for understanding the DPR pathways. The results provide more information about denitrifying polyphosphate-accumulating organisms (DPAOs) in aerobic granular sludge (AGS) and lay the foundations for full-scale DPR.     

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.     

Anaerobic degradation of acetone by Desulfococcus biacutus spec. nov.

From anaerobic digestor sludge of a waste water treatment plant, a gram-negative, strictly anaerobic sulfate-reducing bacterium was isolated with acetone as sole organic substrate. The bacterium was characterized as a new species, Desulfococcus biacutus. The strain grew with acetone with doubling times of 72 h to 120 h; the growth yield was 12.0 (±2.1) g · [mol acetone]^-1. Acetone was oxidized completely, and no isopropanol was formed. In labelling studies with ¹⁴CO₂, cell lipids (including approx. 50% PHB) of acetone-grown cells became labelled 7 times as high as those of 3-hydroxy-buyrate-grown cells. Enzyme studies indicated that acetone was degraded via acetoacetyl-CoA, and that acetone was channeled into the intermediary metabolism after condensation with carbon dioxide to a C₄-compound, possibly free acetoacetate. Acetoacetyl-CoA is cleaved by a thiolase reaction to acetyl-CoA which is completely oxidized through the carbon monoxide dehydrogenase pathway. Strain KMRActS was deposited with the Deutsche Sammlung von Mikroorganismen, Braunschweig, under the number DSM 5651.     

Comparative analysis of hydrogen-producing bacterial biofilms and granular sludge formed in continuous cultures of fermentative bacteria

A system for biohydrogen production was developed based on long-term continuous cultures grown on sugar beet molasses in packed bed reactors. In two separate cultures, consortia of fermentative bacteria developed as biofilms on granitic stones. In one of the cultures, a granular sludge was also formed. Metagenomic analysis of the microbial communities by 454-pyrosequencing of amplified 16S rDNA fragments revealed that the overall biodiversity of the hydrogen-producing cultures was quite small. The stone biofilm from the culture without granular sludge was dominated by Clostridiaceae and heterolactic fermentation bacteria, mainly Leuconostocaeae. Representatives of the Leuconostocaeae and Enterobacteriaceae were dominant in both the granules and the stone biofilm formed in the granular sludge culture. The culture containing granular sludge produced hydrogen significantly more effectively than that containing only the stone biofilm: 5.43 vs. 2.8 mol H₂/mol sucrose from molasses, respectively. The speculations that lactic acid bacteria may favor hydrogen production are discussed.     

Degradation of Methanethiol by Methylotrophic Methanogenic Archaea in a Lab-Scale Upflow Anaerobic Sludge Blanket Reactor

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30°C to H₂S, CO₂, and CH₄. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter⁻¹ day⁻¹. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.     

Enzymatic degradation of granular potato starch by <Emphasis Type="Italic">Microbacterium aurum</Emphasis> strain B8.A

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules.     

Microstructure of Anaerobic Granules Bioaugmented with Desulfitobacterium frappieri PCP-1

Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day⁻¹ and a low bacterial diffusion of 10⁻⁷ dm² day⁻¹. Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.     

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.     

Diversity, Localization, and Physiological Properties of Filamentous Microbes Belonging to Chloroflexi Subphylum I in Mesophilic and Thermophilic Methanogenic Sludge Granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55°C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.