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     

Relationship between Methanogenic Cofactor Content and Maximum Specific Methanogenic Activity of Anaerobic Granular Sludges

In this study we investigated whether a relationship exists between the methanogenic activity and the content of specific methanogenic cofactors of granular sludges cultured on different combinations of volatile fatty acids in upflow anaerobic sludge blanket or fluidized-bed reactors. Significant correlations were measured in both cases between the contents of coenzyme F₄₂₀−2 or methanopterin and the maximum specific methanogenic activities on propionate, butyrate, and hydrogen, but not acetate. For both sludges the content of sarcinapterin appeared to be correlated with methanogenic activities on propionate, butyrate, and acetate, but not hydrogen. Similar correlations were measured with regard to the total content of coenzyme F₄₂₀−4 and F₄₂₀−5 in sludges from fluidized-bed reactors. The results indicate that the contents of specific methanogenic cofactors measured in anaerobic granular sludges can be used to estimate the hydrogenotrophic or acetotrophic methanogenic potential of these sludges.     

Scanning electron microscopy of biofilm development in anaerobic fixed-bed reactors: Influence of the inoculum

Summary Scanning electron microscopy was applied to evaluate the influence of inoculum on efficiency of initial biofilm formation and reactor performance. Five anaerobic fixed-bed reactors were inoculated with anaerobic sludges from different sources and operated in parallel under identical conditions with defined wastewater and acetate, propionate and butyrate as constituents In all sludges Methanothrix sp. was the predominant acetotroph. The reactors inoculated with anaerobic sludge adapted to the wastewater achieved the highest space loading with 21.0 g COD/l·d after 58 days. The inoculation with granular sludge from an upflow anaerobic sludge blanket (UASB) reactor resulted in significantly less reactor efficiency. Time course of biofilm formation and biofilm thickness (ranging from 20–200 m) depended on the type of inoculum.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

Granulation and immobilisation of methanogenic and sulfate-reducing bacteria in high-rate anaerobic reactors

The formation of anaerobic granular sludge on a sulfate-containing waste-water was studied in up-flow anaerobic sludge blanket reactors. Three systems were examined: a sulfidogenic system, a methanogenic system and a mixed sulfidogenic/methanogenic system. No significant granulation was observed in the sulfidogenic system. For the methanogenic and the mixed methanogenic/sulfidogenic system granulation proceeded well, and no significant difference in the granule diameter could be detected. In the three systems studied, different types of sludge developed. A (mainly) methanogenic granular sludge was developed in the methanogenic system, a (more) sulfate-reducing granular sludge was developed in the mixed methanogenic/sulfidogenic system, and a flocculant sulfate-reducing sludge was developed in the sulfidogenic system. Correspondence to: A Visser     

The influence of sulphate on substrate utilization in a thermophilic sewage sludge digestor

Summary Bacterial sulphate reduction and the interaction between sulphate reduction and methane production was studied in an unadapted and sulphate-adapted thermophilic anaerobic sludge digestor. Addition of sulphate to a concentration of 5 mm (100 times the background level) did not influence gas production or volatile fatty acid concentration compared to the control digestor. When sulphate reduction was not limited by the sulphate concentration, the sulphate-adapted digestor had a sulphate reduction rate of 910 mol l^–1 day compared with 17 mol l^–1 day in the control digestor. The results indicate that the potential for sulphate reduction is low in a thermophilic sewage sludge digestor receiving a low sulphate concentration. Counts of sulphate-reducing bacteria and methanogens showed that sulphate-reducing bacteria were found only in significant numbers in the sulphate-adapted digestor and only with H₂/CO₂ as substrate. Only low numbers of acetate-utilizing sulphate-reducing bacteria were found in both digestors. When using radio-labelled acetate, the relative percentage of 2-labelled acetate converted to CO₂ was two to four times higher in the sulphate-adapted digestor compared to the control digestor. These results suggest that oxidation of acetate seems to play a larger role in the sulphate-adapted digestor.Offprint requests to: B. K. Ahring     

Preservation methods for the storage of anaerobic sludges

Different preservation methods were evaluated for the storage of anaerobic sludges: room temperature, refrigeration at 4 °C, freezing at –20 °C and freeze-drying. Specific methanogenic activity for glucose and acetate were used as indicators of the subsequent recovery of the anaerobic sludge. Storage at room temperature and refrigeration resulted in a better conservation of the methanogenic activity than freezing and freeze-drying.     

Anaerobic degradation of volatile fatty acids at different sulphate concentrations

The effect of sulfate on the anaerobic breakdown of mixtures of acetate, propionate and butyrate at three different sulfate to fatty acid ratios was studied in upflow anaerobic sludge blanket reactors. Sludge characteristics were followed with time by means of sludge activity tests and by enumeration of the different physiological bacterial groups. At each sulfate concentration acetate was completely converted into methane and CO₂, and acetotrophic sulfate-reducing bacteria were not detected. Hydrogenotrophic methanogenic bacteria and hydrogenotrophic sulfate-reducing bacteria were present in high numbers in the sludge of all reactors. However, a complete conversion of H₂ by sulfate reducers was found in the reactor operated with excess sulfate. At higher sulfate concentrations, oxidation of propionate by sulfate-reducing bacteria became more important. Only under sulfate-limiting conditions did syntrophic propionate oxidizers out-compete propionate-degrading sulfate reducers. Remarkably, syntrophic butyrate oxidizers were well able to compete with sulfate reducers for the available butyrate, even with an excess of sulfate. Correspondence to: A. Visser     

Anaerobic digestion and wastewater treatment systems

Upflow Anaerobic Sludge Bed (UASB) wastewater (pre-)treatment systems represent a proven sustainable technology for a wide range of very different industrial effluents, including those containing toxic/inhibitory compounds. The process is also feasible for treatment of domestic wastewater with temperatures as low as 14–16° C and likely even lower. Compared to conventional aerobic treatment systems the anaerobic treatment process merely offers advantages. This especially is true for the rate of start-up. The available insight in anaerobic sludge immobilization (i.e. granulation) and growth of granular anaerobic sludge in many respects suffices for practice. In anaerobic treatment the immobilization of balanced microbial communities is essential, because the concentration of intermediates then can be kept sufficiently low.So far ignored factors like the death and decay rate of organisms are of eminent importance for the quality of immobilized anaerobic sludge. Taking these factors into account, it can be shown that there does not exist any need for phase separation when treating non- or slightly acidified wastewaters. Phase separation even is detrimental in case the acidogenic organisms are not removed from the effluent of the acidogenic reactor, because they deteriorate the settleability of granular sludge and also negatively affect the formation and growth of granular sludge. The growing insight in the role of factors like nutrients and trace elements, the effect of metabolic intermediates and end products opens excellent prospects for process control, e.g. for the anaerobic treatment of wastewaters containing mainly methanol.Anaerobic wastewater treatment can also profitably be applied in the thermophilic and psychrophilic temperature range. Moreover, thermophilic anaerobic sludge can be used under mesophilic conditions.The Expanded Granular Sludge Bed (EGSB) system particularly offers big practical potentials, e.g. for very low strength wastewaters (COD 1 g/l) and at temperatures as low as 10° C. In EGSB-systems virtually all the retained sludge is employed, while compared to UASB-systems also a substantially bigger fraction of the immobilized organisms (inside the granules) participates in the process, because an extraordinary high substrate affinity prevails in these systems. It looks necessary to reconsider theories for mass transfer in immobilized anaerobic biomass.Instead of phasing the digestion process, staging of the anaerobic reactors should be applied. In this way mixing up of the sludge can be significantly reduced and a plug flow is promoted. A staged process will provide a higher treatment efficiency and a higher process stability. This especially applies for thermophilic systems.     

Acetate treatment in 70°C upflow anaerobic sludge-blanket (UASB) reactors: start-up with thermophilic inocula and the kinetics of the UASB sludges

This study focused on the use of thermophilic anaerobic granulae in the start-up of 70°C acetate-fed upflow anaerobic sludge-blanket (UASB) reactors and the kinetics of granulae grown at 70°C. In the UASB reactors, chemical oxygen demand removal commenced within 48 h of the start-up. The maximum reduction in chemical oxygen demand was 84% with the feed containing yeast and 71% without a yeast supplement. In the bioassays, the yeast-grown sludge converted 98% of the acetate consumed to methane as compared to 92% for the sludge grown without yeast. The highest initial specific methane production rate (µ_{CH 4}) of the UASB sludges grown at 70°C was 0.088 h^–1 at an acetate concentration of 4.6 mM. The higher initial acetate concentration was found to prolong the lag-phase in methane production significantly and to decrease the µ_{CH 4}. The half-saturation constant (K _s), the inhibition constant (K _i), the inhibition response coefficient (n), and the µ_{CH 4} ^max, calculated according to a modified Haldane equation, were 1.5 mM, 2.8 mM, 0.8, and 0.28 h^–1, respectively. The prolonged starvation of the 70°C sludge (15 days) decreased the µ_{CH 4} from about 0.022 h^–1 to 0.011 h^–1 and increased the lag phase in methane production from 6 h to 24 h as compared to non-starved sludge.     

Acetate and hydrogen metabolism in intact and disintegrated granules from an acetate-fed, 55° C,UASB reactor

Summary To investigate mass-transfer resistance in granules, the effect of disintegration on the specific methanogenic activity (SMA) of acetate-grown anaerobic, thermophilic (55°C) granules was measured. Four different methods of disintegration were used; vortex mixing, ultra-sound, blending and repeated syrine aspiration. When H₂/CO₂ was used as the substrate, disintegrated granules showed a higher SMA than intact granules. However, with acetate as substrate, no effect was observed when granules disintegrated using a vortex mixer or ultra-sound, whereas both the blender- and syringe-treated granules had lower SMAs compared to intact granules. An effectiveness factor, , the ratio of the SMA of disintegrated granules to the SMA of intact granules, was presented and found useful for describing the effectiveness of disintegration to relieve mass-transfer limitation on the granules. Offprint requests to: B. K. Ahring     

Previously unclassified bacteria dominate during thermophilic and mesophilic anaerobic pre-treatment of primary sludge

Thermophilic biological pre-treatment enables enhanced anaerobic digestion for treatment of wastewater sludges but, at present, there is limited understanding of the hydrolytic–acidogenic microbial composition and its contribution to this process. In this study, the process was assessed by comparing the microbiology of thermophilic (50–65 °C) and mesophilic (35 °C) pre-treatment reactors treating primary sludge.     

High tolerance of methanogens in granular sludge to oxygen

This research assessed the effect of oxygen exposure on the methanogenic activity of anaerobic granular sludges. The toxicity of oxygen to acetoclastic methanogens in five different anaerobic granular sludges was determined in serum flasks with effective gas-to-liquid volumes of 4.65 to 1. The amount of oxygen that caused 50% inhibition of the methanogenic activity after 3 days of exposure ranged from 7% to 41% oxygen in the head space. These results indicate that methanogens located in granular sludge have a high tolerance for oxygen. The most important factor contributing to the tolerance was the oxygen consumption by facultative bacteria metabolizing biodegradable substrates. Uptake of oxygen by these bacteria creates anaerobic microenvironments where the methanogenic bacteria are protected. The results also indicate that methanogens in sludge consortia still have some tolerance to oxygen, even in the absence of facultative substrate for oxygen respiration. (c) 1993 John Wiley & Sons, Inc.     

Effect of seed sludge microbial community and activity on the performance of anaerobic reactors during the start-up period

In this study, two laboratory-scale anaerobic batch reactors started up with different inoculum sludges and fed with the same synthetic wastewater were monitored in terms of performance and microbial community shift by denaturant gradient gel electrophoresis fingerprinting and subsequent cloning, sequencing analysis in order to reveal importance of initial quality of inoculum sludge for operation of anaerobic reactors. For this purpose, two different seed sludge were evaluated. In Reactor1 seeded with a sludge having less diverse microbial community (19 operational taxonomic unit (OTU’s) for Bacterial and 8 OTU’s for Archaeal community, respectively) and a methanogenic activity of 150 ml CH₄ g TVS⁻¹ day⁻¹, a chemical oxygen demand (COD) removal efficiency of 78.8 ± 4.17% was obtained at a substrate to microorganism (S/X) ratio of 0.38. On the other hand, Reactor2, seeded with a sludge having a much more diverse microbial community (24 OTU’s for Bacterial and 9 OTU’s for Archaeal communities, respectively) and a methanogenic activity, 450 ml CH₄ g TVS⁻¹ day⁻¹, operated in the same conditions showed a better start-up performance; a COD removal efficiency of over 98% at a S/X ratio of 0.53. Sequence analysis of Seed2 revealed the presence of diverse fermentative and syntrophic bacteria, whereas excised bands of Seed1 related to fermentative and sulfate/metal-reducing bacteria. This study revealed that a higher degree of bacterial diversity, especially the presence of syntrophic bacteria besides the abundance of key species such as methanogenic Archaea may play an important role in the performance of anaerobic reactors during the start-up period.     

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.     

Methanotroph and methanogen coupling in granular biofilm under O2-limited conditions

Summary The co-culture between Methylosinus sporium, a strictly aerobic methanotroph, and strictly anaerobic methanogens was studied in 5 L aerobic/anaerobic coupled granular sludge reactors under O₂-limited conditions. The methanogenic bacteria maintained very good metabolic activities and were able to produce sufficient methane which serviced as substrate for methanotrophic growth. Although other strictly aerobic population proliferated by two orders of magnitude after the granular sludge had been operated under O₂-limited conditions for one month, only a limited amount of the added methanotroph remained in the sludge. This result may indicate that M. sporium lacks sufficient O₂ affinity to compete with facultative bacteria for the dissolved O₂ for their growth.     

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with ¹⁴C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with ¹³C₆-glucose and ¹³C₃-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with ¹³C-glucose and ¹³C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with ¹⁴C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K_m for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5–10 m). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.     

Selenomonas acidaminovorans sp. nov., a versatile thermophilic proton-reducing anaerobe able to grow by decarboxylation of succinate to propionate

A moderately thermophilic anaerobic bacterium (strain Su883), which decarboxylated succinate to propionate, was isolated from granular methanogenic sludge. The bacterium appeared to ferment a number of amino acids including glutamate, histidine, arginine, ornithine, citrulline, and threonine to propionate, acetate and hydrogen. Propionate was formed via the oxidative decarboxylation of -ketoglutarate to succinyl-CoA. In addition, the strain degraded glucose, fructose, glycerol, pyruvate, serine, alanine, citrate and malate to acetate, carbon dioxide and hydrogen, and branched-chain amino acids to branched-chain fatty acids. With all single substrates solely hydrogen was formed as reduced fermentation product. Mixed cultures of strain Su883 and Methanobacterium thermoautotrophicum H showed a more rapid conversion of substrates and with some substrates a shift from acetate to propionate formation.Strain Su883 is a motile, gram-negative, non-sporeforming, slightly curved rod with a DNA base ratio of 56.5 mol% guanine-plus-cytosine. Selenomonas acidaminovorans Su883 is proposed as type strain for the new species within the genus Selenomonas.     

Illumination enhances methane production from thermophilic anaerobic digestion

Incandescent lamp illumination enhanced methane production from a thermophilic anaerobic digestion reactor (55°C) supplied with glucose. After 10 days of operation, the volume of methane produced from light reactors was approximately 2.5 times higher than that from dark reactors. A comparison of the carbon balance between light and dark conditions showed that methane produced from hydrogen and carbon dioxide in the light reactors was higher than that from the dark reactors. When hydrogen or acetate was fed into the reactors, methane production with added hydrogen was faster and higher under light conditions than under dark conditions. The use of blue light-emitting diodes also enhanced methane production over that under dark conditions. The 16S rRNA gene copy numbers for Methanothermobacter spp. in the light reactor and in the dark reactor were at the same level. The copy number for Methanosarcina spp. in the light reactors was approximately double than that in the dark reactors. These results suggest that blue light enhances the methanogenic activity of hydrogenotrophic methanogens.     

13C-NMR Study of propionate metabolism by sludges from bioreactors treating sulfate and sulfide rich wastewater

Applications of nuclear magnetic resonance (NMR) to study a variety of physiological and biochemical aspects of bacteria with a role in the sulfur cycle are reviewed. Then, a case-study of high resolution¹³ C-NMR spectroscopy on sludges from bioreactors used for treating sulfate and sulfide rich wastewaters is presented.¹³ C-NMR was used to study the effect of sulfate and butyrate on propionate conversion by mesophilic anaerobic (methanogenic and sulfate reducing) granular sludge and microaerobic (sulfide oxidizing) flocculant sludge. In the presence of sulfate, propionate was degraded via the randomising pathway in all sludge types investigated. This was evidenced by scrambling of [3-¹³C]propionate into [2-¹³C]propionate and the formation of acetate equally labeled in the C₁ and C₂ position. In the absence of sulfate, [3-¹³C]propionate scrambled to a lesser extend without being degraded further. Anaerobic sludges converted [2,3-¹³C]propionate partly into the higher fatty acid 2-methyl[2,3-¹³C]butyrate during the simultaneous degradation of [2,3-¹³C]propionate and butyrate. [4,5-¹³C]valerate was also formed in the methanogenic sludges. Up to 10% of the propionate present was converted via these alternative degradation routes. Labeled butyrate was not detected in the incubations, suggesting that reductive carboxylation of propionate does not occur in the sludges.