Contact Angle Measurement and Cell Hydrophobicity of Granular Sludge from Upflow Anaerobic Sludge Bed Reactors

The contact angle, which is generally used to evaluate the hydrophobicities of pure bacterial strains and solid surfaces, was used to study mixed cell cultures of bacteria involved in anaerobic digestion. Previously published data and data from this study showed that most acidogens are hydrophilic (contact angle, <45(deg)) but most of the acetogens and methanogens isolated from granular sludge are hydrophobic (contact angle, >45(deg)). The hydrophobicities of mixtures of hydrophilic and hydrophobic cells were found to be linearly correlated with the cell mixing ratio. The hydrophobicities of cells present in effluents from upflow anaerobic sludge bed reactors which were treating different types of substrates were different depending on the reactor conditions. When the reactor liquid had a high surface tension, cells sloughing off from sludge granules, as well as cells present on the outer surfaces of the granules, were hydrophobic. Short-term batch enrichment cultures revealed that proteins selected for highly hydrophilic cells. Long-term in-reactor enrichment cultures revealed that sugars selected for hydrophilic acidogens on the surfaces of the granules, while fatty acids tended to enrich for hydrophobic methanogens. When linear alkylbenzenesulfonate was added, the cells on the surfaces of granules became more hydrophilic. Control tests performed with pure cultures revealed that there was no change in the surface properties due to linear alkylbenzenesulfonate; hence, the changes in the wash-out observed probably reflect changes in the species composition of the microbial association. A surface layer with moderate hydrophobicity, a middle layer with extremely high hydrophobicity, and a core with high hydrophobicity could be distinguished in the grey granules which we studied.     

Improved Dechlorinating Performance of Upflow Anaerobic Sludge Blanket Reactors by Incorporation of Dehalospirillum multivorans into Granular Sludge

Dechlorination of tetrachloroethene, also known as perchloroethylene (PCE), was investigated in an upflow anaerobic sludge blanket (UASB) reactor after incorporation of the strictly anaerobic, reductively dechlorinating bacterium Dehalospirillum multivorans into granular sludge. This reactor was compared to the reference 1 (R1) reactor, where the granules were autoclaved to remove all dechlorinating abilities before inoculation, and to the reference 2 (R2) reactor, containing only living granular sludge. All three reactors were fed mineral medium containing 3 to 57 μM PCE, 2 mM formate, and 0.5 mM acetate and were operated under sterile conditions. In the test reactor, an average of 93% (mole/mole) of the effluent chloroethenes was dichloroethene (DCE), compared to 99% (mole/mole) in the R1 reactor. The R2 reactor, with no inoculation, produced only trichloroethene (TCE), averaging 43% (mole/mole) of the effluent chloroethenes. No dechlorination of PCE was observed in an abiotic control consisting of sterile granules without inoculum. During continuous operation with stepwise-reduced hydraulic retention times (HRTs), both the test reactor and the R1 reactor showed conversion of PCE to DCE, even at HRTs much lower than the reciprocal maximum specific growth rate of D. multivorans, indicating that this bacterium was immobilized in the living and autoclaved granular sludge. In contrast, the R2 reactor, with no inoculation of D. multivorans, only converted PCE to TCE under the same conditions. Immobilization could be confirmed by using fluorescein-labeled antibody probes raised against D. multivorans. In granules obtained from the R1 reactor, D. multivorans grew mainly in microcolonies located in the centers of the granules, while in the test reactor, the bacterium mainly covered the surfaces of granules.     

Acetate Degradation at 70 degrees C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70 degrees C

Anaerobic acetate degradation at 70 degrees C and at 55 degrees C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70 degrees C and less than 15 days was needed at 55 degrees C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55 degrees C up to 90% of the COD was removed. Batch assays showed that sludges from two 70 degrees C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70 degrees C. Both 70 degrees C sludges, as well as the 55 degrees C sludge, produced methane at temperatures of 37 to 73 degrees C. The 55 degrees C sludge exhibited shorter lag phases than the 70 degrees C sludges and higher specific methane production rates between 37 and 65 degrees C.     

Phenotypic Properties and Microbial Diversity of Methanogenic Granules from a Full-Scale Upflow Anaerobic Sludge Bed Reactor Treating Brewery Wastewater

Methanogenic granules from an anaerobic bioreactor that treated wastewater of a beer brewery consisted of different morphological types of granules. In this study, the microbial compositions of the different granules were analyzed by molecular microbiological techniques: cloning, denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH), and scanning and transmission electron microscopy. We propose here that the different types of granules reflect the different stages in the life cycle of granules. Young granules were small, black, and compact and harbored active cells. Gray granules were the most abundant granules. These granules have a multilayer structure with channels and void areas. The core was composed of dead or starving cells with low activity. The brown granules, which were the largest granules, showed a loose and amorphous structure with big channels that resulted in fractured zones and corresponded to the older granules. Firmicutes (as determined by FISH) and Nitrospira and Deferribacteres (as determined by cloning and sequencing) were the predominant Bacteria. Remarkably, Firmicutes could not be detected in the brown granules. The methanogenic Archaea identified were Methanosaeta concilii (70 to 90% by FISH and cloning), Methanosarcina mazei, and Methanospirillum spp. The phenotypic appearance of the granules reflected the physiological condition of the granules. This may be valuable to easily select appropriate seed sludges to start up other reactors.     

Microbial and Physicochemical Characteristics of Compact Anaerobic Ammonium-Oxidizing Granules in an Upflow Anaerobic Sludge Blanket Reactor

Anaerobic ammonium oxidation (anammox) is a promising new process to treat high-strength nitrogenous wastewater. Due to the low growth rate of anaerobic ammonium-oxidizing bacteria, efficient biomass retention is essential for reactor operation. Therefore, we studied the settling ability and community composition of the anaerobic ammonium-oxidizing granules, which were cultivated in an upflow anaerobic sludge blanket (UASB) reactor seeded with aerobic granules. With this seed, the start-up period was less than 160 days at a NH₄⁺-N removal efficiency of 94% and a loading rate of 0.064 kg N per kg volatile suspended solids per day. The formed granules were bright red and had a high settling velocity (41 to 79 m h⁻¹). Cells and extracellular polymeric substances were evenly distributed over the anaerobic ammonium-oxidizing granules. The high percentage of anaerobic ammonium-oxidizing bacteria in the granules could be visualized by fluorescent in situ hybridization and electron microscopy. The copy numbers of 16S rRNA genes of anaerobic ammonium-oxidizing bacteria in the granules were determined to be 4.6 × 10⁸ copies ml⁻¹. The results of this study could be used for a better design, shorter start-up time, and more stable operation of anammox systems for the treatment of nitrogen-rich wastewaters.The anaerobic ammonia oxidation (anammox) process is a recently discovered biological nitrogen removal technology in which ammonia is oxidized to nitrogen gas with nitrite as the electron acceptor (5, 29, 32). In contrast to heterotrophic denitrification (6, 26), the anammox process does not require external electron donors (e.g., methanol) due to their chemolithoautotrophic lifestyle. Furthermore, if this process is combined with a partial nitrification step, only half of the ammonium needs to be nitrified to nitrite, which together with the remaining ammonium can subsequently be converted into nitrogen through the anammox process. This reduces the oxygen demand of the system and leads to further reduction in operational costs (27).The anaerobic ammonium-oxidizing bacteria (anammox bacteria) have a low growth rate (18), with a doubling time at best estimated as 7 to 11 days (18, 28). The yield of the anammox bacteria has been determined to be 0.066 mol C biomass mol⁻¹ ammonium consumed, and the maximum ammonium consumption rate is ∼45 nmol mg⁻¹ protein min⁻¹ (18). Given the low growth rate and low yield, very efficient biomass retention is essential to retain the anammox bacteria within the reactor systems during cultivation (19). The enrichment of anammox bacteria from a mixed inoculum requires the optimization of conditions favorable for the anammox bacteria and generally takes 200 to 300 days (5, 6, 27). Thus, conditions that would reduce the start-up time of anammox reactors would positively effect the implementation of the process. Several sources of inocula, such as activated sludge (4), nitrifying activated sludge (27), and anaerobic sludge (6), have been used for the start-up of anammox reactors with start-up times of as long as 1,000 days (27).Aerobic granules have been reported to have high microbial diversity (31) and compact structure with very good settling properties resulting in an efficient means of biomass retention. These properties, including interspecies competition and mass transfer, result in the stratification of microbial species with anoxic pockets in the interior of the granules that may be suitable to harbor anammox bacteria. Therefore, the main objective of this study was to investigate the feasibility of start-up of the anammox process by seeding the reactor with aerobic granular sludge by using an upflow anaerobic sludge blanket (UASB) reactor. After the successful start-up and the formation of anammox granules, the structure and physicochemical properties of the anammox granules and the reactor performance were characterized. Microbial community analysis revealed that the dominant anammox species was related to a species of anammox bacteria present in anammox biofilms.     

UASB-化学混凝技术处理草浆造纸废水的研究

采用UASB-化学混凝技术组合对草浆造纸废水进行处理。结果表明,接种颗粒污泥的二级启动,大大缩短了反应器的启动时间。在水力停留时间(HRT)6 h,进水COD逐渐提高的情况下对颗粒污泥进行驯化,20 d后pH、COD去除率趋于稳定。另外,通过L9(34)正交试验,研究了混凝中4因子影响大小的排序,并在最优条件下对UASB反应器出水进行混凝处理。组合技术处理的出水水质指标符合国家造纸工业污水排放标准。     

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.     

Start-up criteria for a Upflow Anaerobic Sludge Blanket (UASB) reactor

A laboratory scale Upflow Anaerobic Sludge Blanket (UASB) reactor study was done with spentwash (distillery effluent) as substrate. The start-up studies revealed that the recirculation of biogas, produced during the initial stages, under low organic loading rates helps in mixing as well as partial supplementation of carbon dioxide for excess hydrogen in the reactor. The gas composition before and after recirculation, the volatile acids profile and the solids variation along the reactor height before and after recirculation were studied. The process efficiency and methane content increases substantially after recirculation of biogas and the microscopic examination of the sludge during the experiment indicates that the granules mainly composed of long multicellular filaments of rod shaped organisms, presumably methanothrix soehngenii.     

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.     

Methanogenesis in an Upflow Anaerobic Sludge Blanket Reactor at pH 6 on an Acetate-Propionate Mixture

High-rate anaerobic digestion can be applied in upflow anaerobic sludge blanket reactors for the treatment of various wastewaters. In upflow anaerobic sludge blanket reactors, sludge retention time is increased by a natural immobilization mechanism (viz. the formation of a granular type of sludge). When this sludge is cultivated on acid-containing wastewater, the granules mainly consist of an acetoclastic methanogen resembling Methanothrix soehngenii. This organism grows either in rods or in long filaments. Attempts to cultivate a stable sludge consisting predominantly of Methanosarcina sp. on an acetate-propionate mixture as substrate by lowering the pH from 7.5 during the start-up to approximately 6 failed. After 140 days of continuous operation of the reactor a filamentous organism resembling Methanothrix soehngenii prevailed in the sludge. The specific methanogenic activity of this sludge on acetate-propionate was optimal at pH 6.6 to 6.8 and 7.0 to 7.2, respectively.     

Start‐up of Anaerobic Hydrogen Producing Reactors Seeded with Sewage Sludge

The procedure for starting‐up continuously stirred tank reactors (CSTR) for acclimating anaerobic hydrogen‐producing microorganisms with sewage sludge was investigated. Initially, feeding with glucose and sucrose as well as mixing were carried out in semicontinuous mode; hydraulic retention time (HRT) was in an order of 20, 15, 10, 5, 2.5 and 2 days. When the pH declined to its lowest value (pH 5.18), it was adjusted to 6.7 using sodium hydroxide (1 N). At the same time, the semi‐continuous operation was changed to a continuous one. Finally, the pH was continuously regulated at approximately 6.7. The results indicate that this procedure can be used to cultivate seed sludge for hydrogen production from sewage sludge resulting in a large hydrogen production in less than 60 days. When the substrate was glucose, a hydrogen yield of 1.63 mol H₂/mol glucose and a specific hydrogen production rate of 321 mmol H₂/g VSS day at an HRT of 13.3 h was achieved. When the substrate was sucrose with the same HTR, a hydrogen yield of 4.45 mol H2/mol sucrose and a specific hydrogen production rate of 707 mmol H₂/g VSS day was obtained.     

Acetate Degradation at 70°C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70°C

Anaerobic acetate degradation at 70°C and at 55°C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70°C and less than 15 days was needed at 55°C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55°C up to 90% of the COD was removed. Batch assays showed that sludges from two 70°C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70°C. Both 70°C sludges, as well as the 55°C sludge, produced methane at temperatures of 37 to 73°C. The 55°C sludge exhibited shorter lag phases than the 70°C sludges and higher specific methane production rates between 37 and 65°C.     

Enhancement of Sulphide Production in Anaerobic Packed Bed Bench-scale Biofilm Reactors by Sulphate Reducing Bacteria

Two biofilm reactors, using pumice stone and Poraver as biofilm supports, were run, based on the optimization of sulphide production using a factorial design. The maximum H₂S concentrations reached were 10 and 15 mm, respectively, both being appropriate for metal precipitation in effluents. The set-up of the pumice stone biofilm reactor is suitable for application in the mining area in the Bolivian Andean region, where this material is widely available. The use of specific primers for sulphate-reducing bacteria groups permits the identification of the sulphide-producing bacteria present in biofilms.     

Cluster Structure of Anaerobic Aggregates of an Expanded Granular Sludge Bed Reactor

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH₄/g of volatile suspended solids [VSS]·day or 1.1 g of CH₄ chemical oxygen demand/g of VSS·day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.     

Methanogens revealed immunologically in granules from five Upflow Anaerobic Sludge Blanket (UASB) bioreactors grown on different substrates

Abstract Methanogens were identified and quantified using antibody probes and the antigenic fingerprinting method in five different kinds of granular sludge taken from five Uplow Anaerobic Sludge Blanket (UASB) bioreactors maintained on different substrates. The methanogenic flora present in each bioreactor was elucidated and expressed in cells per garm dry weight. Autofluorescence, phase-contrast and bright field-microscopy of unstained and Gram-stained preparations were used in parallel with immunotechnology to characterize each methanogenic subpopulation. Ten different methanogens were prevalent in the five bioreactor systems. Methanogens antigenically related to Methanobacterium formicicum MF, Methanobrevibacter arboriphilus AZ and Methanothrix soehngenii Opfikon were found in all five granules, while other methanogens were present in only some. A trend was observed towards a wider diversity of methanogenic subpopulations parallelling an increase in the complexity of the bioreactor's substrate.     

Fluidized Bed Hot-Melt Granulation as a Tool to Improve Curcuminoid Solubility

Curcumin is the main bioactive component of Curcuma longa L. and has recently aroused growing interest from the scientific community. Unfortunately, the medicinal properties attributed to curcuminoids are impaired by their low oral bioavailability or low solubility in aqueous solutions. Many strategies have been studied to improve curcumin solubility; however, the preparation of granules using hydrophilic materials has never been attempted. The aim of this work was to develop curcumin granules by fluidized bed hot-melt granulation using the hydrophilic carrier Gelucire® 50:13. A two-level factorial design was used to verify the influence of Gelucire® 50:13 and lactose contents found in the granules on their size, morphology, bulk and tapped densities, flow, moisture content, and water activity. The granules obtained were also evaluated by differential scanning calorimetry, thermogravimetric analysis, X-ray powder diffraction, and infrared spectrometry. The curcumin solubility and dissolution rates in water were determined by liquid chromatography. The best formulation provides an increase of curcumin solubility of 4642-fold and 3.8-fold compared to the physical mixture. The dissolution tests showed a maximum drug release from granules after 45 min of 70% at pH 1.2 and 80% at pH 5.8 and 7.4, while for non-granulated curcumin, the release was below 20% in all pH. The solid-state characterization and solubility measurement showed good stability of granules over 9 months. The results attest that the fluidized bed hot-melt granulation with hydrophilic binders is an attractive and promising alternative to obtain solid forms of curcumin with enhanced bioavailability.     

Desulfovibrio mexicanus sp. nov., a Sulfate-reducing Bacterium Isolated from an Upflow Anaerobic Sludge Blanket (UASB) Reactor Treating Cheese Wastewaters

A mesophilic sulfate-reducing bacterium, designated strain Lup1^T(T=type strain) was isolated from a Mexican UASB digester treating cheese factory wastewater. The non-motile, Gram-negative, curved and non-spore-forming cells (1.7–2.5×0.5 μm) existed singly or in chains. Optimum growth occurred at 37°C and pH 7.2 in a medium containing lactate and thiosulfate. Strain Lup1^Tused pyruvate, formate, Casamino acids, serine, cysteine, H₂and ethanol as electron donors in the presence of thiosulfate as an electron acceptor and fermented pyruvate, Casamino acids, cysteine, and serine. Sulfate, elemental sulfur, and sulfite also served as electron acceptors but not nitrate or fumarate. Thiosulfate was disproportionated to sulfate and sulfide. The G+C content of the DNA was 66 mol%. Phylogenetic analysis based on 16S rDNA revealed that strain Strain Lup1^Twas a member of the genus Desulfovibrio withDesulfovibrio aminophilus being the closest relative (similarity value of 91%). As strain Lup1^Tis physiologically and phylogenetically different from other Desulfovibrio species, it is designated Desulfovibrio mexicanus sp. nov. (=DSM 13116).     

Direct Characterization of Methanogens in Two High-Rate Anaerobic Biological Reactors

The methanogenic flora from two types of turbulent, high-rate reactors was studied by immunologic methods as well as by phase-contrast, fluorescence, and scanning electron microscopy. The reactors were a fluidized sand-bed biofilm ANITRON reactor and an ultrafiltration membrane-associated suspended growth MARS reactor (both trademarks of Air Products and Chemicals, Inc., Allentown, Pa.). Conventional microscopic methods revealed complex mixtures of microbes of a range of sizes and shapes, among which morphotypes resembling Methanothrix spp. and Methanosarcina spp. were noticed. Precise identification of these and other methanogens was accomplished by antigenic fingerprinting with a comprehensive panel of calibrated antibody probes of predefined specificity spectra. The methanogens identified showed morphotypes and antigenic fingerprints indicating their close similarity with the following reference organisms: Methanobacterium formicicum MF and Methanosarcina barkeri W in the ANITRON reactor only; Methanosarcina barkeri R1M3, M. mazei S6, Methanogenium cariaci JR1, and Methanobrevibacter arboriphilus AZ in the MARS reactor only; and Methanobrevibacter smithii ALI and Methanothrix soehngenii Opfikon in both reactors. Species diversity and distribution appeared to be, at least in part, dependent on the degree of turbulence inside the reactor.     

Water Surface Energy Contribution to Adherence of Hydrophobic Groups in Relation to Stability of Protein Conformations

I wish to advance a new approach concerning the contribution of water to hydrophobic interactions, namely that the adherence of hydrophobic groups is enhanced by the loss of water surface free energy which arises from the reduction of the surface area of the water originally adjoining the hydrophobic contact areas; and that this energy loss can be computed in the following way.