Isolation and characterization of Veillonella sp. from methanogenic granular sludge

An anaerobic, propionate-producing, mesophilic, Gram-negative, non-spore forming, non-motile, coccoid-shaped bacterium (strain S119) was isolated from methanogenic granular sludge of an upflow anaerobic sludge blanket reactor. Based on morphology and cytological and physiological properties the isolate was assigned to the genus Veillonella. Strain S119 forms spherical monospecies biofilms (granules), 1.0–3.0 mm in diameter, when grown in continuously mixed medium with sodium lactate as the sole carbon source and powdered activated carbon as biofilm support particles. The granules attained concentrations of volatile suspended solids up to 38 mg/cm³. Veillonella sp. strain S119 has a highly hydrophobic cell surface and produces extracellular slime, which contains polysaccharide fractions. Growth characteristics and adhesion properties of the isolated microorganisms suggest its participation in the formation of granular sludge. Correspondence to: W. Verstraete     

Diffusional Properties of Methanogenic Granular Sludge: 1H NMR Characterization

The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging. NMR spectroscopy measurements were performed at 22°C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons). Self-diffusion coefficients of H₂O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water. Interestingly, self-diffusion coefficients of H₂O were independent of the aggregate size for the size fractions investigated. Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55°C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10°C) temperatures. Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90°C for 30 min) modified the diffusional transport up to 20%. In contrast, deactivation of aggregates by HgCl₂ did not affect the H₂O self-diffusion coefficient in aggregates. Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.     

Gratuitous dechlorination of chloroethanes by methanogenic granular sludge

The dechlorinating activity of a methanogenic granular sludge from a methanol-fed upflow anaerobic sludge blanket reactor was investigated with chlorinated ethanes. This unadapted methanogenic consortium degraded all chloroethanes tested. The product formation rates decreased with the number of chlorine substituents. The more highly chlorinated ethanes were also converted, although at a lower rate, in the presence of autoclaved (dead) sludge, indicating the involvement of reduced heat-stable cofactors like vitamin B₁₂ and F₄₃₀. Direct chemical dechlorination of hexa-, penta- and tetrachloroethanes was also observed in medium without sludge, although at a much lower rate. The results show the importance of cometabolic and abiotic (chemical) conversions for the transformation of chlorinated ethanes by the methanogenic consortium. The types of reaction and the products formed were correlated with the Gibbs free-energy change (ΔG ^0′). Reductive hydrogenolysis and dichloroelimination were important dechlorinating mechanisms. Generally, these reactions have a higher ΔG ^0′ value than dehydrochlorination reactions, which occurred less frequently during the transformation of chloroethanes by the methanogenic granular sludge. Received: 8 June 1998 / Received revision: 7 September 1998 / Accepted: 13 September 1998     

Localization and quantification of extracellular polymers in methanogenic granular sludge

Summary Extracellular polymers were localized and quantitatively analysed in methanogenic granular sludge cultivated on either propionate or ethanol in laboratory upflow anaerobic sludge-blanket (UASB) reactors. Electron microscopical analysis of ultrathin sections of the two sludge types stained with ruthenium red revealed the presence of extracellular polymers with different densities and structures. For quantification, granular sludge from a large-scale UASB reactor at a liquid sugar plant was also included in this study. A three-step physical disintegration procedure was used to extract water-soluble extracellular material from the granules. After each disintegration step the extracts were analysed for polysaccharides and proteins. Cell damage and thus the contribution of intracellular proteins and polysaccharides was estimated simultaneously by the determination of free DNA and free ATP in the extracts. After two extraction steps, up to 3.5 mg polysaccharides/g organic material and 5.5 mg protein/g organic material were extracted, whereas no significant increase in DNA was detected. The role of extracellular polymers in granular stability is discussed. Offsprint requests to: A. J. B. Zehnder     

Rapid acclimation of methanogenic granular sludge into denitrifying sulfide removal granules

Rapid formation of denitrifying sulfide removal granules is of practical interest to start up an expanded granular sludge bed reactor for wastewater treatment. This study demonstrates that methanogenic granules can be easily acclimated into DSR granules in one day, removing all 1.30 kg m⁻³ d⁻¹ sulfide and converting >90% of 0.56 kg-N m⁻³d⁻¹ nitrate into di-nitrogen gas. Under high loadings, reactor performance, however, declined. Under high loading rates, sulfide first inhibited the heterotrophic denitrifier (Caldithrix sp.), thereby accumulating nitrite in the system; the autotrophic denitrifier (Pseudomonas sp. C23) was then inhibited by accumulated nitrite, leading to breakdown of the entire DSR process.     

Anaerobic degradation of benzaldehyde in methanogenic granular sludge: the influence of additional substrates

Summary The degradation of benzaldehyde in methanogenic granular sludge was investigated in batch and in upflow anaerobic sludge blanket (UASB) reactors. The effect due to the presence of co-substrates, such as H₂, sodium butyrate and sucrose, was studied using formaldehyde as a reference compound. The additional substrates enhanced the activity of benzaldehyde- and formaldehyde-degrading microorganisms (ACT_bdm and ACT_fdm, respectiveky) and increased the transient production of benzyl alcohol and methanol. As a consequence, the concentrations of benzaldehyde and formaldehyde that caused 50% inhibition of the methanogenic activity (50% IC_m) on sucrose were 3133 and 254 mg chemical oxygen demand (COD)/l respectively, three times higher than the literature data values on acetate. Experiments performed in UASB reactors on benzaldehyde showed that the replacement of volatile fatty acids with sucrose as co-substrate improved the treatment capacity of the system from 0.73 to 4.36 kg COD benzaldehyde·m^–13·day^–1. Correspondence to: O. Todini     

Toxicity of N-substituted aromatics to acetoclastic methanogenic activity in granular sludge.

N-substituted aromatics are important priority pollutants entering the environment primarily through anthropogenic activities associated with the industrial production of dyes, explosives, pesticides, and pharmaceuticals. Anaerobic treatment of wastewaters discharged by these industries could potentially be problematical as a result of the high toxicity of N-substituted aromatics. The objective of this study was to examine the structure-toxicity relationships of N-substituted aromatic compounds to acetoclastic methanogenic bacteria. The toxicity was assayed in serum flasks by measuring methane production in granular sludge. Unacclimated cultures were used to minimize the biotransformation of the toxic organic chemicals during the test. The nature and the degree of the aromatic substitution were observed to have a profound effect on the toxicity of the test compound. Nitroaromatic compounds were, on the average, over 500-fold more toxic than their corresponding aromatic amines. Considering the facile reduction of nitro groups by anaerobic microorganisms, a dramatic detoxification of nitroaromatics towards methanogens can be expected to occur during anaerobic wastewater treatment. While the toxicity exerted by the N-substituted aromatic compounds was closely correlated with compound apolarity (log P), it was observed that at any given log P, N-substituted phenols had a toxicity that was 2 orders of magnitude higher than that of chlorophenols and alkylphenols. This indicates that toxicity due to the chemical reactivity of nitroaromatics is much more important than partitioning effects in bacterial membranes.     

Enrichment and granulation of Anammox biomass started up with methanogenic granular sludge

A sequencing batch reactor (SBR) seeded with methanogenic granular sludge was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the feasibility of granulation of Anammox biomass. Research results showed that hydraulic retention time (HRT) was an important factor to enrich Anammox bacteria. When the HRT was controlled at 30 days during the initial cultivation, the SBR reactor presented Anammox activity at t = 58 days. Simultaneously, the methanogenic granular sludge changed gradually from dust black to brown colour and its diameter became smaller. At t = 90 days, the Anammox activity was further improved. NH₄⁺-N and NO₂⁻N were removed simultaneously with higher speed and the maximum removal rates reached 14.6 g NH₄⁺-N /(m³ _reactor·day) and 6.67 g NO₂⁻-N /(m³ _reactor·day), respectively. Between t = 110 days and t = 161 days, the nitrogen load was increased to a HRT of 5 days (70 mg/l NH₄⁺ and 70 mg/l NO₂), the removal rates of ammonium and nitrite were 60.6% and 62.5% respectively. The sludge changed to red and formed Anammox granulation with high nitrogen removal activity.     

Characteristics of granular methanogenic sludge grown on glucose in a UASB reactor

The formation of granules grown on glucose in an upflow anaerobic sludge blanket (UASB) reactor was investigated. Total granular sludge concentration retained in the UASB reactor was 34.5 g MLSS/l (30.0 g MLVSS/l) during 240 d operation on glucose minimum medium with the supplementation of 1.07 g NaHCO₃ per 1 g glucose. This realized a high-rate methanogenic fermentation of glucose of 17.6 g COD/l-reactor-d at 3.4 d⁻¹ of space velocity. The granules formed were relatively small, ranging mainly from 0.4 to 0.5 mm, had a relatively low cell density of 0.0542–0.0560 g MLVSS/ml, and had low specific gravity (0.97–1.19) due to very low ash content (11–13%). Electron microscopic analysis showed that Methanothrix spp. appeared dominant over the granules. The specific metabolic activities of bacterial trophic groups were the highest for H₂ followed by glucose, acetate, and propionate.     

Effect of chlorinated aliphatic hydrocarbons on the acetoclastic methanogenic activity of granular sludge

The toxicity of chlorinated aliphatic hydrocarbons on acetoclastic methanogens in anaerobic granular sludge was determined using a standardized anaerobic bioassay method. Most of the chloroaliphatics tested were strong inhibitors of methanogenesis. Tri- and tetrachloride derivatives of methane and ethane were the most highly toxic compounds tested, with concentrations of less than 18 mg/l resulting in 50% inhibition (IC₅₀) of the methanogenic activity. Dichlorinated compounds were less toxic, with IC₅₀ values ranging from 40 mg/l to 100 mg/l. On the other hand, perchlorinated derivatives of ethane and ethene were scarcely inhibitory at concentrations near their maximum water solubility. The toxicity caused by chlorinated aliphatic hydrocarbons was reversible. The comparison of structurally related compounds indicated that unsaturated chloroaliphatics were less toxic than their saturated counterparts. A reverse correlation between the electric dipole moment of these compounds and their methanogenic toxicity is discussed. Received: 9 July 1996 / Received revision: 11 October 1996 / Accepted: 18 October 1996     

Effect of nickel deprivation on methanol degradation in a methanogenic granular sludge bioreactor

The effect of omitting nickel from the influent on methanol conversion in an Upflow Anaerobic Sludge Bed (UASB) reactor was investigated. The UASB reactor (30°C, pH 7) was operated for 261 days at a 12-h hydraulic retention time (HRT) and at organic loading rates (OLRs) ranging from 2.6 to 7.8 g COD l reactor⁻¹ day⁻¹. The nickel content of the sludge decreased by 66% during the 261-day reactor run because of washout and doubling of the sludge bed volume. Nickel deprivation initially had a strong impact on the methanogenic activity of the sludge with methanol; e.g., after 89 days of operation, this activity was doubled by adding 2 μM nickel. Upon prolonged UASB reactor operation, methanol and VFA effluent concentrations decreased whereas the sludge lost its response to nickel addition in activity tests. This suggests that a less nickel-dependent methanol-converting sludge had developed in the UASB reactor. Received 09 April 2002/ Accepted in revised form 13 July 2002     

High rate performance and characterization of granular methanogenic sludges in upflow anaerobic sludge blanket reactors fed with various defined substrates

High rate granular methanogenic fermentations were performed in one-phase upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewaters containing starch, sucrose, ethanol, and butyrate plus propionate. All granules formed showed high settling velocities which enabled high cell mass retention and accommodation of high loading rates. The maximum COD removal rates (g COD/l-reactor·d) obtained after 500-d operations were 7.6 for starch, 10.5 for sucrose, 32.1 for ethanol, and 42.6 for butyrate-propionate. Long-term growth on various defined substrates altered the population of bacterial trophic groups and overall characteristics of granules. The starch- and sucrose-grown granules were characterized by larger size and more abundant extracellular polymeric substances (EPS) than the ethanol- or fatty acids-grown granules. The fatty acids-grown granules contained a considerable amount of inorganic salts (ash content: 56 to 63%) but a small amount of EPS, and showed a denser ultrastructure than the other three types of granules. The granules grown on ethanol under slightly acidic conditions showed the lowest specific gravity and volatile suspended solids (VSS) density as well as ash content among all of the granules. As aceticlastic methanogens, Methanothrix spp. were predominant in the starch-, sucrose-, and fatty acids-grown granules, whereas comparable numbers of Methanosarcina spp. were observed only in the ethanol-grown granules. The populations of hydrogenotrophic methanogens were the largest of all bacterial trophic groups in the respective granules. The data confirm that the prevalence of Methanothrix spp. and high methanogenic activity for H₂ are general characteristics of methanogenic granucles and that EPS and inorganic deposits contribute chemically to the enhancement of structural stability and mechanical strength of granules.     

Characteristics of granular methanogenic sludge grown on phenol synthetic medium and methanogenic fermentation of phenolic wastewater in a UASB reactor

The growth of granules on a phenol synthetic medium and the methanogenic fermentation of industrial phenolic wastewater from a steel factory in an upflow anaerobic sludge blanket (UASB) reactor were investigated. Total granular sludge concentration retained in the UASB reactor was 6.7 g MLSS/l (6.0 g MLVSS/l) during the 10 months' operation on the phenol synthetic medium. This realized a maximum phenol removal rate of 2.2 g/l·d (phenol concentration of influent = 500 mg/l), which corresponded to 5.2 g COD/l·d at space velocity (SV) of 4.4 d⁻¹. The granules formed were of relatively small size ranging from 0.61 to 0.77 mm, and had a relatively low density of 0.013–0.023 g MLVSS/cm³ and low specific gravity (1.11) due to very low ash content (8.7–11.9%). Electron microscopic analysis showed that Methanothrix spp. appeared dominantly on the granule surface as well as within it. The specific metabolic activities of bacterial trophic groups were the highest for H₂ followed by acetate, benzoate, phenol, and propionate. In the case of industrial phenolic wastewater, although phenol efficiency was only 50% at SV of 0.4 d⁻¹, when the wastewater was diluted twofold and the treated wastewater was recycled at SV of 7.3 d⁻¹, the removal efficiencies of phenol and CODcr were restored to 90% (influent=400 mg/l) and 80% (influent=5,000 mg/l), respectively. It was suggested that recycling of the treated wastewater might be improved by partly degrading unknown toxic compounds contained in phenolic wastewater.     

Inorganic composition and microbial characteristics of methanogenic granular sludge grown in a thermophilic upflow anaerobic sludge blanket reactor

An upflow anaerobic sludge blanket reactor was operated under thermophilic conditions (55° C) for 160 days by feeding a wastewater containing sucrose as the major carbon source. The reactor exhibited a satisfactory performance due to the formation of well-settling granulated sludge, achieving a total organic carbon (TOC) removal of above 80% at an organic loading rate of 30 kg total organic C m^–3 day^–1. Structural and microbial properties of the methanogenic granular sludge were examined using scanning electron microscope X-ray analyses and serum vial activity tests. All the thermophilic granules developed showed a double-layered structure, comprised of a black core portion and a yellowish exterior portion. The interior cope portion contained abundant crystalline precipitates of calcium carbonate. Calcium-bound phosphorus was also present more prominently in the core portion than in the exterior portion. Methanogenic activities of the thermophilic granules both from acetate and from H₂ increased with increasing vial-test temperature in the range of 55–65° C [from 1.43 to 2.36 kg CH₄ chemical oxygen demand (COD) kg volatile suspended solids (VSS)^–1 day^–1 for acetate and from 0.85 to 1.11 kg CH₄ COD kg VSS^–1 day^–1 for H₂]. On the other hand, propionate-utilizing methanogenic activity was independent of vial-test temperature, and was much lower (0.1–0.12 kg CH₄ COD kg VSS^–1 day^–1) than that from either acetate or H₂. Acetate consumption during vial tests was considerably inhibited by the presence of H₂ in the headspace, indicating that a syntrophic association between acetate oxidizers and H₂-utilizing methane-producing bacteria was responsible for some portion of the overall acetate elimination by the theromophilically grown sludge.     

Effect of long-term cobalt deprivation on methanol degradation in a methanogenic granular sludge bioreactor

The effect of the trace metal cobalt on the conversion of methanol in an upflow anaerobic sludge bed (UASB) reactor was investigated by studying the effect of cobalt deprivation from the influent on the reactor efficiency and the sludge characteristics. A UASB reactor (30 degrees C; pH 7) was operated for 261 days at a 12-h hydraulic retention time (HRT). The loading rate was increased stepwise from 2.6 g chemical oxygen demand (COD) x L reactor(-1) x d(-1) to 7.8 g COD x L reactor(-1) x d(-1). Cobalt deprivation had a strong impact on the methanogenic activity of the sludge. In batch tests, the methanogenic activity of the sludge with methanol as the substrate increased 5.3 (day 28) and 2.1 (day 257) times by addition of 840 nM of cobalt. The sludge had an apparent K(m) for cobalt of 948 nM after 28 days of operation and 442 nM at the end of the run. Cobalt deprivation during 54 days of operation led to a methanol conversion efficiency of only 55%. Continuous addition of cobalt (330 nM) for 33 days improved the methanol removal efficiency to 100%. In this period of cobalt dosing, the cobalt concentration in the sludge increased 2.7 times up to 32 microg x g TSS(-1). Upon omission of the cobalt addition, cobalt washed-out at a stable rate of 0.1 microg x g VSS(-1) x d(-1). At the end of the run, the cobalt concentration of the sludge was similar to that of the seed sludge.     

1H NMR characterization of two crambin species

Crambin displays amino acid heterogeneity at positions 22 (Pro or Ser) and 25 (Leu or Ile). Using reversed phase HPLC the crambin mixture can be resolved into two protein fractions. It is shown by amino acid analysis and NMR spectroscopy that these fractions represent single proteins (Ser-22/Ile-25 and Pro-22/ Leu-25 species). A first characterization of the ¹H-NMR spectra of these species is presented.     

1H NMR relaxometric characterization of bovine lactoferrin

Lactoferrin (Lf) is a mammalian iron binding protein present in external secretions and in polymorphonuclear leukocytes. Its role in host defense mechanisms related to the non-immune defense system has been definitively established. Lf has two identical iron-binding sites, far from each other (44.3 A) and magnetically non-interacting. Fe(III) ions are six-coordinated, with four donor atoms provided by protein sidechains (two Tyr, one His, one Asp) and two oxygen atoms from a bridged HCO(3)(-). This set of ligands provides an ideal coordination scheme for stable and reversible iron binding. Nuclear magnetic relaxation dispersion (NMRD) profiles of Lf are consistent with a closest distance for a single water hydrogen atom of 3.1 A. By looking at the X-ray structure of Lf (PDB ID code: 1BLF) we can locate two water oxygens at 3.95 and 4.27 A from each Fe(III), respectively. Temperature dependence data suggest that an important contribution to the overall paramagnetic contribution to the solvent water relaxation rate arises from one or more second sphere water molecules in slow exchange with the bulk. A decreasing value of the exchange rate is obtained, ranging from 1.2 to 0.7 micros in the observed temperature range (25-65 degrees C), with an activation enthalpy of 7.3+/-0.8 kJ mol(-1). The low exchange rate obtained from NMRD data can be explained by the observation that both water molecules are bound to several polar groups of the protein backbone and side chains. By increasing the pH from 6.5 to 12 two distinct titrations are observed, consistent with sequential removal of both water molecules.     

Population dynamics of propionate-oxidizing bacteria under methanogenic and sulfidogenic conditions in anaerobic granular sludge.

Laboratory-scale upflow anaerobic sludge-bed reactors were inoculated with industrial granular sludge and fed with either propionate or propionate and sulfate. The population dynamics of the propionate-oxidizing bacteria Desulfobulbus sp. and the syntrophically growing strain SYN7 were studied in reactors by dot blot and in situ hybridization with 16S rRNA-based oligonucleotide probes.     

Physicochemical characteristics of anaerobic H2-producing granular sludge

Granule-based biological H₂ production processes are gaining great popularity in recent years. An efficient and stable operating of such systems relies heavily on the performance of the H₂-producing granules (HPGs), which possess many unique properties compared with floc sludge and methanogenic granules. Hence, a full understanding of the sludge characteristics is essential. Especially, the physicochemical properties of HPGs may provide useful information for effective evaluation of system status. This review offers a systematical introduction of the physicochemical properties of HPGs, including size, morphology, settling velocity, permeability, rheology, surface charge, hydrophobicity and extracellular polymeric substances (EPS). We also analyze the relationships between these physicochemical factors and the system performance, and discuss the remaining challenges and future implications for sludge characterization and process monitoring. This work may facilitate a better understanding of granule-based biological H₂ production processes and offer a basis for timely process monitoring and manipulation.