Assessment of metabolic properties and kinetic parameters of methanogenic sludge by on-line methane production rate measurements

This report presents a new approach to studying the metabolic and kinetic properties of anaerobic sludge from single batch experiments. The two main features of the method are that the methane production is measured on-line with a relatively cheap system, and that the methane production data can be plotted as rate vs time curves. The case studies of specific methanogenic activity, biodegradability and toxicity tests here presented show that very accurate kinetic data can be obtained. The method is specifically useful in experiments in which strong changes in methane production occur, and it is proposed as a powerful tool to study methanogenic systems. Furthermore, the method is simple and could be implemented by industry in the routine analysis of sludge.     

Structure of coenzyme F(420) dependent methylenetetrahydromethanopterin reductase from two methanogenic archaea

Coenzyme F(420)-dependent methylenetetrahydromethanopterin reductase (Mer) is an enzyme of the Cl metabolism in methanogenic and sulfate reducing archaea. It is composed of identical 35-40 kDa subunits and lacks a prosthetic group. The crystal structure of Mer from Methanopyrus kandleri (kMer) revealed in one crystal form a dimeric and in another a tetrameric oligomerisation state and that from Methanobacterium thermoautotrophicum (tMer) a dimeric state. Each monomer is primarily composed of a TIM-barrel fold enlarged by three insertion regions. Insertion regions 1 and 2 contribute to intersubunit interactions. Insertion regions 2 and 3 together with the C-terminal end of the TIM-barrel core form a cleft where the binding sites of coenzyme F(420) and methylene-tetrahydromethanopterin are postulated. Close to the coenzyme F(420)-binding site lies a rarely observed non-prolyl cis-peptide bond. It is surprising that Mer is structurally most similar to a bacterial FMN-dependent luciferase which contains a non-prolyl cis-peptide bond at the equivalent position. The structure of Mer is also related to that of NADP-dependent FAD-harbouring methylenetetrahydrofolate reductase (MetF). However, Mer and MetF do not show sequence similarities although they bind related substrates and catalyze an analogous reaction.     

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.     

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.     

寺河矿煤地质产甲烷微生物菌群的保藏和产甲烷性能

【背景】煤地质产甲烷微生物菌群可以代谢煤基质产生甲烷,对于实现煤层气资源的再利用具有重要意义。【目的】检测产甲烷菌群在保藏过程中群落结构的动态变化以及在产气实验中甲烷气的生成情况,以验证保藏方法的可行性,同时为煤层气的微生物增产奠定基础。【方法】分别于不同温度条件下比较3种菌种保藏方法,即甘油/L-半胱氨酸法、富营养法和煤基-基础盐法。通过产气实验检测不同保藏条件下产甲烷菌群的活力。同时,采用454高通量测序技术测定16S r RNA基因序列,分析25°C条件下煤基-基础盐菌种保藏过程中微生物群落结构的变化。【结果】比较了9组菌种保藏方法,发现菌种最佳保藏条件为25°C的煤基-基础盐保藏。在该条件下保藏的产甲烷菌群活性最高,甲烷生成量最大。以无烟煤为碳源进行产气实验时甲烷生成量为12%-25%,而以褐煤为碳源时甲烷生成量可达24%-73%。在25°C的煤基-基础盐菌种保藏条件下,保藏初期细菌的主要优势菌为假单胞菌属(Pseudomonas),而古菌的主要优势菌为甲烷八叠球菌属(Methanosarcina)。随着保藏时间的增加,细菌的群落结构变化显著,发酵细菌及产氢产乙酸细菌成为优势细菌,古菌的群落结构则相对稳定。【结论】菌种保藏的最佳条件为25°C的煤基-基础盐,保藏的产甲烷菌群能长期维持在较高的活性状态,具有较好的产甲烷能力。     

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.     

Biochemical methane potential (BMP) test for thickened sludge using anaerobic granular sludge at different inoculum/substrate ratios

The biochemical methane potential (BMP) test for thickened sludge was evaluated at three different inoculum/substrate (I:S) ratios. The cumulative methane yield was 51.4 mL CH₄/g VS_added at an I:S ratio of 1:1, 76.3 mL CH₄/g VS_added at an I:S ratio of 1:3, and 21.9 mL CH₄/g VS_added at an I:S ratio of 1:8. The greatest ultimate methane yield and methane production rate constant were achieved at an I:S ratio of 1:3, whereas the least was obtained at an I:S ratio of 1:8. The maximum methane production rate constant was 0.38/day and the minimum methane production rate constant was 0.0016/day. For the case of a lower I:S ratio, the biomass activity may be affected due to the low substrate concentration. On the other hand, for the case of higher I:S ratios, anaerobic digestion of thickened sludge was inhibited by higher concentrations of volatile fatty acids and lower pH.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

Quantitation of coenzyme F420 in methanogenic sludge by the use of reversed-phase high-performance liquid chromatography and a fluorescence detector

Summary With the use of acetone extraction, reversed-phase High-Performance Liquid Chromatography and fluorimetric monitoring, the quantity of coenzyme F₄₂₀ in mixed liquors and rumen contents can be measured. A synthetic analog of coenzyme F₄₂₀ is used as an internal standard to compensate for differences in fluorimetric monitoring. The method allows the detection of one picomol of coenzyme F₄₂₀ and the differentiation between different forms of the coenzyme known to be present in various methanogenic bacteria.     

Extracellular polymers in granular sludge from different upflow anaerobic sludge blanket (UASB) reactors

Thermal extraction was used to quantify extracellular polymers (ECP) in granules from anaerobic upflow reactors. The optimal time for extraction was determined as the time needed before the intracellular material gives a significant contribution to the extracted extracellular material due to cell lysis. ECP contents of 41 to 92 mg · g^–1 volatile suspended solids of granules were found depending on the type of granular sludge examined. The content of polysaccharides, protein and lipids in the extracted ECP was quantified. Furthermore, the different methyl esters of the lipids were determined and quantified. Lower amounts of polysaccharides and proteins were found in the extracellular material from granules grown on methanogenic and acetogenic substrates compared to granules grown on more complex substrates. In contrast, the lipid content was lower on complex substrates. Changing the feed of an upflow anaerobic sludge blanket reactor from a sugar-containing waste-water to a synthetic waste-water containing acetate, propionate and butyrate resulted in a decrease in both the protein and polysaccharide content and an increase in the lipid content of the extracellular material. Furthermore, the amount of protein and polysaccharides in the ECP found under mesophilic conditions was significantly higher than under thermophilic conditions, while the lipid content was lower.     

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     

Microbial characteristics of methanogenic sludge consortia developed in thermophilic U ASB reactors

The effect of temperature on granulation and microbial interaction of anaerobic sludges grown in thermophilic upflow anaerobic sludge bed (UASB) reactors was investigated at two different temperatures, 55°C (Run 1) and 65°C (Run 2). Each run consisted of two phases. Phase 1 was conducted by feeding acetate for a period of 200 days. In Phase 2, both reactors were fed a mixture of acetate and sucrose for a further 100 days. During Phase 1, no granulation occurred in the sludge of either run. Microscopic observation revealed that the predominant methanogen was Methanothrix in Run 1, whereas Methanobacterium-like bacteria existed to a significant extent in Run 2. The acetate-utilizing methanogenic activity of both sludges increased with increasing test temperature in the range 55–65°C. Since the acetate-grown sludges exhibited far higher H₂-utilizing methanogenic activity than acetate-utilizing methanogenic activity, it is suggested that a syntrophic association of acetate-oxidizing bacteria with hydrogenotrophic methanogens was responsible for a considerable portion of the overall acetate elimination in thermophilic anaerobic sludge. During Phase 2, granules coated with either filamentous bacteria or cocci-type bacteria (both presumably acid-forming bacteria) were successfully established in Run 1 and Run 2, respectively. Since the acetate-utilizing methanogenic activities of the granular sludges were four to five times higher than those of the acetate-grown sludges (Phase 1), the co-existence of these coating bacteria appeared to contribute to the enclosing of acetate consumers inside granules. Correspondence to: S. Uemura     

Anaerobic treatment of protein-containing waste waters: correlation between coenzyme F420 and methane production

Summary Anaerobic treatment of gelatine-containing model waste water and baker's yeast manufacturing effluent was investigated in upflow anaerobic sludge blanket (UASB) reactors. During start up a correlation between coenzyme F ₄₂₀ content and methane production in the reactor was observed. By monitoring coenzyme F ₄₂₀ concentrations a certain prediction of methanogenic activities was possible.     

Role of nickel in high rate methanol degradation in anaerobic granular sludge bioreactors

The effect of nickel deprivation from the influent of a mesophilic (30 degrees C) methanol fed upflow anaerobic sludge bed (UASB) reactor was investigated by coupling the reactor performance to the evolution of the Methanosarcina population of the bioreactor sludge. The reactor was operated at pH 7.0 and an organic loading rate (OLR) of 5-15 g COD l(-1) day(-1) for 191 days. A clear limitation of the specific methanogenic activity (SMA) on methanol due to the absence of nickel was observed after 129 days of bioreactor operation: the SMA of the sludge in medium with the complete trace metal solution except nickel amounted to 1.164 (+/-0.167) g CH(4)-COD g VSS(-1) day(-1) compared to 2.027 (+/-0.111) g CH(4)-COD g VSS(-1) day(-1) in a medium with the complete (including nickel) trace metal solution. The methanol removal efficiency during these 129 days was 99%, no volatile fatty acid (VFA) accumulation was observed and the size of the Methanosarcina population increased compared to the seed sludge. Continuation of the UASB reactor operation with the nickel limited sludge lead to incomplete methanol removal, and thus methanol accumulation in the reactor effluent from day 142 onwards. This methanol accumulation subsequently induced an increase of the acetogenic activity in the UASB reactor on day 160. On day 165, 77% of the methanol fed to the system was converted to acetate and the Methanosarcina population size had substantially decreased. Inclusion of 0.5 muM Ni (dosed as NiCl(2)) to the influent from day 165 onwards lead to the recovery of the methanol removal efficiency to 99% without VFA accumulation within 2 days of bioreactor operation.     

Quantification of coenzyme F420 analogues from methanogenic bacteria by HPLC and fluorimetry

Summary A quantitative assay for analogues of coenzyme F₄₂₀ is presented. The assay combines separation of the coenzymes by binary reversed-phase high performance liquid chromatography with detection by fluorescence, yielding high specificity and sensitivity. Quantification is by calibration with a coenzyme F₄₂₀ standard or by employing coenzyme F₄₂₀ fragments as internal standards.     

Kinetics and mass-transfer phenomena in anaerobic granular sludge

The kinetic properties of acetate-degrading methanogenic granular sludge of different mean diameters were assessed at different up-flow velocities (V(up)). Using this approach, the influence of internal and external mass transfer could be estimated. First, the apparent Monod constant (K(S)) for each data set was calculated by means of a curve-fitting procedure. The experimental results revealed that variations in the V(up) did not affect the apparent K(S)-value, indicating that external mass-transport resistance normally can be neglected. With regard to the granule size, a clear increase in K(S) was found at increasing granule diameters. The experimental data were further used to validate a dynamic mathematical biofilm model. The biofilm model was able to describe reaction-diffusion kinetics in anaerobic granules, using a single value for the effective diffusion coefficient in the granules. This suggests that biogas formation did not influence the diffusion-rates in the granular biomass.     

Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge

Summary Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above. Offprint requests to: J. B. van Lier     

Diversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor

The ammonium-oxidizing microbial community was investigated in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor that was operated for about 1 year with high anaerobic ammonium oxidation activity (up to 0.8 kg NH(4)(+)-N m(-3) day(-1)). A Planctomycetales-specific 16S rRNA gene library was constructed to analyse the diversity of the anaerobic ammonium-oxidizing bacteria (AnAOB). Most of the specifically amplified sequences (15/16) were similar to each other (> 99%) but were distantly related to all of the previously recognized sequences (< 94%), with the exception of an unclassified anammox-related clone, KSU-1 (98%). An ammonia monooxygenase (amoA) gene library was also analysed to investigate the diversity of 'aerobic' ammonium-oxidizing bacteria (AAOB) from the beta-Proteobacteria. Most of the amoA gene fragments (53/55) clustered in the Nitrosomonas europaea-Nitrosococcus mobilis group which has been reported to prevail under oxygen-limiting conditions. The quantitative results from real-time polymerase chain reaction (PCR) amplification showed that the dominant AnAOB comprised approximately 50% of the total bacterial 16S rRNA genes in the reactor, whereas the AAOB of beta-Proteobacteria represented only about 3%. A large fragment (4008 bp) of the rRNA gene cluster of the dominant AnAOB (AS-1) in this reactor sludge was sequenced and compared with sequences of other Planctomycetales including four anammox-related candidate genera. The partial sequence of hydrazine-oxidizing enzyme (hzo) of dominant AnAOB was also identified using new designed primers. Based on this analysis, we propose to tentatively name this new AnAOB Candidatus'Jettenia asiatica'.