The potential of osmolytes and their precursors to alleviate osmotic stress of anaerobic granular sludge.

Increasing amounts of saline (waste)water with high concentrations of organic pollutants are generated globally. In the anaerobic (waste)water treatment domain, high salt concentrations are repeatedly reported to inhibit methanogenic activity and strategies to overcome this toxicity are needed. Current research focuses on the use of potential osmolyte precursor compounds for osmotic stress alleviation in granular anaerobic sludges upon exposure to hypersalinity shocks. Glutamic acid, aspartic acid, lysine, potassium, gelatine, and tryptone were tested for their potential to alleviate osmotic stress in laboratory grown and full - scale granular sludge. The laboratory grown granular sludge was adapted to 5 (R5) and 20 (R20) g Na+/L. Full-scale granular sludge was obtained from internal circulation reactors treating tannery (waste)water with influent conductivity of 29.2 (Do) and 14.1 (Li) mS/cm. In batch experiments which focused on specific methanogenic activity (SMA), R5 granular sludge was exposed to a hypersalinity shock of 20 g Na+/L. The granular sludge of Do and Li was exposed to a hypersalinity shock of 10 g Na+/L with sodium acetate as the sole carbon source. The effects on R20 granular sludge were studied at the salinity level to which the sludge was already adapted, namely 20 g Na+/L. Dosing of glutamic acid, aspartic acid, gelatine, and tryptone resulted in increased SMA compared to only acetate fed batches. In batches with added glutamic acid, the SMA increased by 115% (Li), 35% (Do) and 9% (R20). With added aspartic acid, SMA increased by 72% (Li), 26% (Do), 12% (R5) and 7% (R20). The addition of tryptone resulted in SMA increases of 36% (R5), 17% (R20), 179% (Li), and 48% (Do), whereas added gelatine increased the SMA by 30% (R5), 14% (R20), 23% (Li), and 13% (Do). The addition of lysine, meanwhile, gave negative effects on SMA of all tested granular sludges. Potassium at sea water Na/K ratio (27.8 w/w) had a slight positive effect on SMA of Do (7.3%) and Li (10.1%), whereas at double the sea water ratio (13.9% w/w) had no pronounced positive effect. R20 granular sludge was also exposed to hyposalinity shock from 20 down to 5 g Na+/L. Glutamate and N-acetyl-ß-lysine were excreted by microbial consortium in anaerobic granular sludge adapted to 20 g Na+/L upon this exposure to hyposalinity. A potential consequence when applying these results is that saline streams containing specific and hydrolysable proteins can be anaerobically treated without additional dosing of osmolytes.

Fast anaerobic sludge granulation at elevated salinity.

It is commonly accepted that high salt concentrations negatively affect microbial activity in biological wastewater treatment reactors such as upflow anaerobic sludge blanket (UASB) reactors. Microbial aggregation in such reactors is equally important. It is well documented that anaerobic granules, when exposed to high salinity become weak and disintegrate, causing wash-out, operational problems and decreasing process performance. In this research, the possibility of microbial granule formation from dispersed biomass was investigated at salinity levels of 5 and 20 g Na+/L. High removal efficiencies of soluble influent organics were achieved at both salinity levels and this was accompanied by fast and robust formation of microbial granules. The process was found to be stable for the entire operational period of 217 days. As far as we know this is the first time it has been demonstrated that stable granule formation is possible at a salinity level as high as 20 g Na+/L. Methanosaeta was identified as the dominant methanogen at both salinity levels. Streptococcus spp. and bacteria belonging to the family Lachnospiraceae were identified as the dominant microbial population at 5 and 20 and g Na+/L, respectively.

Read-across of ready biodegradability based on the substrate specificity of N-alkyl polypropylene polyamine-degrading microorganisms.

The biodegradation of N-alkyl polypropylene polyamines (NAPPs) was studied using pure and mixed cultures to enable read-across of ready biodegradability test results. Two Pseudomonas spp. were isolated from activated sludge with N-oleyl alkyl propylene diamine and N-coco alkyl dipropylene triamine, respectively. Both strains utilized all NAPPs tested as the sole source of carbon, nitrogen and energy for growth. Mineralization of NAPPs was independent of the alkyl chain length and the size of the polyamine moiety. NAPPs degraded in closed bottle tests (CBTs) using both river water and activated sludge. However, ready biodegradability of NAPPs with alkyl chain lengths of 16-18 carbon atoms and polyamine moieties with three and four nitrogen atoms could not be demonstrated. Biodegradation in the CBT was hampered by their limited bioavailability, making assessment of the true ready biodegradability of these highly adsorptive surfactants impossible. All NAPPs are therefore classified as readily biodegradable through read-across. Read-across is justified by the broad substrate specificity of NAPP-degrading microorganisms, their omnipresence and the mineralization of NAPPs.

Biofilm formation and granule properties in anaerobic digestion at high salinity.

For the anaerobic biological treatment of saline wastewater, Anaerobic Digestion (AD) is currently a possibility, even though elevated salt concentrations can be a major obstacle. Anaerobic consortia and especially methanogenic archaea are very sensitive to fluctuations in salinity. When working with Upflow Sludge Blanket Reactor (UASB) technology, in which the microorganisms are aggregated and retained in the system as a granular biofilm, high sodium concentration negatively affects aggregation and consequently process performances. In this research, we analysed the structure of the biofilm and granules formed during the anaerobic treatment of high salinity (at 10 and 20 g/L of sodium) synthetic wastewater at lab scale. The acclimated inoculum was able to accomplish high rates of organics removal at all the salinity levels tested. 16S rRNA gene clonal analysis and Fluorescence In Situ Hybridization (FISH) analyses identified the acetoclastic Methanosaeta harundinacea as the key player involved acetate degradation and microbial attachment/granulation. When additional calcium (1 g/L) was added to overcome the negative effect of sodium on microbial aggregation, during the biofilm formation process microbial attachment and acetate degradation decreased. The same result was observed on granules formation: while calcium had a positive effect on granules strength when added to UASB reactors, Methanosaeta filaments were not present and the degradation of the partially acidified substrate was negatively influenced. This research demonstrated the possibility to get granulation at high salinity, bringing to the forefront the importance of a selection towards Methanosaeta cells growing in filamentous form to obtain strong and healthy granules.

Characterization of the bacterial community involved in the bioflocculation process of wastewater organic matter in high-loaded MBRs.

High-loaded membrane bioreactors (HL-MBRs), i.e., bioreactors equipped with a membrane for biomass retention and operated at extremely short sludge and hydraulic retention times, can concentrate sewage organic matter to facilitate subsequent energy and chemical recovery from these organics. Bioflocculation, accomplished by microorganisms that produce extracellular polymers, is a very important mechanism in these reactors. Bacterial diversity of the sludge and supernatant fraction of HL-MBRs operated at very short sludge retention times (0.125, 0.5, and 1 day) were determined using a PCR-denaturing gradient gel electrophoresis (DGGE) and clone library approach and compared to the diversity in sewage. Already at a sludge retention time (SRT) of 0.125 day, a distinct bacterial community developed compared to the community in sewage. Bioflocculation, however, was low and the majority of the bacteria, especially Arcobacter, were present in the supernatant fraction. Upon increasing SRT from 0.125 to 1 day, a much stronger bioflocculation was accompanied by an increased abundance of Bacteroidetes in the (solid) sludge fraction: 27.5 % at an SRT of 0.5 day and 46.4 % at an SRT of 1 day. Furthermore, cluster analysis of DGGE profiles revealed that the bacterial community structure in the sludge was different from that in the supernatant. To localize specific bacterial classes in the sludge flocs, fluorescence in situ hybridization (FISH) was carried out with three different bacterial probes. This showed that Betaproteobacteria formed clusters in the sludge flocs whereas Alphaproteobacteria and Gammaproteobacteria were mainly present as single cells.

Moorella stamsii sp. nov., a new anaerobic thermophilic hydrogenogenic carboxydotroph isolated from digester sludge.

A novel anaerobic, thermophilic, carbon monoxide-utilizing bacterium, strain E3-O(T), was isolated from anaerobic sludge from a municipal solid waste digester. Cells were straight rods, 0.6-1 µm in diameter and 2-3 µm in length and grew as single cells or in pairs. Cells formed round terminal endospores. The temperature range for growth was 50-70 °C, with an optimum at 65 °C. The pH range for growth was 5.7-8.0, with an optimum at 7.5. Strain E3-O(T) had the ability to ferment various sugars, such as fructose, galactose, glucose, mannose, raffinose, ribose, sucrose and xylose, producing mainly H2 and acetate. In addition, the isolate was able to grow with CO as the sole carbon and energy source. CO oxidation was coupled to H2 and CO2 formation. The G+C content of the genomic DNA was 54.6 mol%. Based on 16S rRNA gene sequence analysis, this bacterium is most closely related to Moorella glycerini (97 % sequence identity). Based on the physiological features and phylogenetic analysis, it is proposed that strain E3-O(T) should be classified in the genus Moorella as a representative of a novel species, Moorella stamsii. The type strain of Moorella stamsii is E3-O(T) ( = DSM 26271(T) = CGMCC 1.5181(T)).

Metabolic interactions in methanogenic and sulfate-reducing bioreactors.

In environments where the amount of electron acceptors is insufficient for complete breakdown of organic matter, methane is formed as the major reduced end product. In such methanogenic environments organic acids are degraded by syntrophic consortia of acetogenic bacteria and methanogenic archaea. Hydrogen consumption by methanogens is essential for acetogenic bacteria to convert organic acids to acetate and hydrogen. Several syntrophic cocultures growing on propionate and butyrate have been described. These syntrophic fatty acid-degrading consortia are affected by the presence of sulfate. When sulfate is present sulfate-reducing bacteria compete with methanogenic archaea for hydrogen and acetate, and with acetogenic bacteria for propionate and butyrate. Sulfate-reducing bacteria easily outcompete methanogens for hydrogen, but the presence of acetate as carbon source may influence the outcome of the competition. By contrast, acetoclastic methanogens can compete reasonably well with acetate-degrading sulfate reducers. Sulfate-reducing bacteria grow much faster on propionate and butyrate than syntrophic consortia.

Carbon monoxide conversion by thermophilic sulfate-reducing bacteria in pure culture and in co-culture with Carboxydothermus hydrogenoformans.

Biological sulfate (SO(4)) reduction with carbon monoxide (CO) as electron donor was investigated. Four thermophilic SO(4)-reducing bacteria, Desulfotomaculum thermoacetoxidans (DSM 5813), Thermodesulfovibrio yellowstonii (ATCC 51303), Desulfotomaculum kuznetsovii (DSM 6115; VKM B-1805), and Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum (DSM 14055), were studied in pure culture and in co-culture with the thermophilic carboxydotrophic bacterium Carboxydothermus hydrogenoformans (DSM 6008). D. thermoacetoxidans and T. yellowstonii were extremely sensitive to CO: their growth on pyruvate was completely inhibited at CO concentrations above 2% in the gas phase. D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were less sensitive to CO. In pure culture, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were able to grow on CO as the only electron donor and, in particular in the presence of hydrogen/carbon dioxide, at CO concentrations as high as 50-70%. The latter SO(4) reducers coupled CO oxidation to SO(4) reduction, but a large part of the CO was converted to acetate. In co-culture with C. hydrogenoformans, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum could even grow with 100% CO (P(CO) = 120 kPa).

Interspecies electron transfer in methanogenic propionate degrading consortia.

Propionate is a key intermediate in the conversion of complex organic matter under methanogenic conditions. Oxidation of this compound requires obligate syntrophic consortia of acetogenic proton- and bicarbonate reducing bacteria and methanogenic archaea. Although H(2) acts as an electron-carrier in these consortia, evidence accumulates that formate plays an even more important role. To make energy yield from propionate oxidation energetically feasible for the bacteria and archaea involved, the concentrations of H(2) and formate have to be extremely low. On the other hand, the diffusion distance of these carriers has to be small to allow high propionate conversion rates. Accordingly, the high conversion rates observed in methanogenic bioreactors are due to the fact that the propionate-oxidizing bacteria and their methanogenic partners form micro-colonies within the densely packed granules.

Enrichment of thermophilic syntrophic anaerobic glutamate-degrading consortia using a dialysis membrane reactor.

A dialysis cultivation system was used to enrich slow-growing moderately thermophilic anaerobic bacteria at high cell densities. Bicarbonate buffered mineral salts medium with 5 mM glutamate as the sole carbon and energy source was used and the incubation temperature was 55 degrees C. The reactor inoculum originated from anaerobic methanogenic granular sludge bed reactors. The microbial population was monitored over a period of 2 years using the most probable number (MPN) technique. In the reactor glutamate was readily degraded to ammonium, methane, and carbon dioxide. Cell numbers of glutamate-degrading organisms increased 400-fold over the first year. In medium supplemented with bromoethane sulfonic acid (BES, an inhibitor of methanogenesis), tenfold lower cell numbers were counted, indicating the syntrophic nature of glutamate degradation. After 2 years of reactor operation the predominant organisms were isolated and characterized. Methanobacterium thermoautotrophicum (strain R43) and a Methanosaeta thermophila strain (strain A) were the predominant hydrogenotrophic and acetoclastic methanogens, respectively. The numbers in which the organisms were present in the reactor after 24 months of incubation were 8.6 x 10(9) and 3.8 x 10(7) mL(-1) sludge, respectively. The most predominant glutamate-degrading organism (8.6 x 10(7) mL(-1) sludge), strain Z, was identified as a new species, Caloramator coolhaasii. It converted glutamate to hydrogen, acetate, some propionate, ammonium, and carbon dioxide. Growth of this syntrophic organism on glutamate was strongly enhanced by the presence of methanogens.

Elucidation of the pathways of catabolic glutamate conversion in three thermophilic anaerobic bacteria.

The glutamate catabolism of three thermophilic syntrophic anaerobes was compared based on the combined use of [(13)C] glutamate NMR measurements and enzyme activity determinations. In some cases the uptake of intermediates from different pathways was studied. The three organisms, Caloramator coolhaasii, Thermanaerovibrio acidaminovorans and strain TGO, had a different stoichiometry of glutamate conversion and were dependent on the presence of a hydrogen scavenger (Methanobacterium thermoautotrophicum Z245) to a different degree for their growth. C. coolhaasii formed acetate, CO(2), NH(4)(+) and H(2) from glutamate. Acetate was found to be formed through the beta-methylaspartate pathway in pure culture as well as in coculture. T. acidaminovorans converted glutamate to acetate, propionate, CO(2), NH(4)(+) and H(2). Most likely, this organism uses the beta-methylaspartate pathway for acetate formation. Propionate formation occurred through a direct oxidation of glutamate via succinyl-CoA and methylmalonyl-CoA. The metabolism of T. acidaminovorans shifted in favour of propionate formation when grown in coculture with the methanogen, but this did not lead to the use of a different glutamate degradation pathway. Strain TGO, an obligate syntrophic glutamate-degrading organism, formed propionate, traces of succinate, CO(2), NH(4)(+) and H(2). Glutamate was converted to propionate oxidatively via the intermediates succinyl-CoA and methylmalonyl-CoA. A minor part of the succinyl-CoA was converted to succinate and excreted.

Arginine catabolism by Thermanaerovibrio acidaminovorans.

The arginine catabolism of Thermanaerovibrio acidaminovorans was investigated. T. acidaminovorans was able to produce approximately 0.4--0.5 mol citrulline and 0.5--0.6 mol ornithine from 1 mol of arginine. However, in a methanogenic coculture with Methanobacterium thermoautotrophicum Z245 1 mol arginine was converted to approximately 1 mol of propionate, 0.5 mol acetate, 4 mol ammonia and 4 mol hydrogen; citrulline and ornithine were not formed. Enzyme measurements indicated the presence of the arginine deiminase pathway (ADI) in cells of T. acidaminovorans growing on arginine.

Caloramator coolhaasii sp. nov., a glutamate-degrading, moderately thermophilic anaerobe.

An obligately anaerobic, moderately thermophilic, glutamate-degrading bacterium (strain ZT) was isolated from an enrichment culture obtained from anaerobic thermophilic granular sludge. The cells were rod-shaped to filamentous and showed no motility or spore formation. The cell wall had a Gram-positive structure, which was revealed by electron microscopy. Optimum growth of the strain was observed under neutrophilic conditions at 50-55 degrees C. The doubling time of strain ZT grown in rich medium was approximately 1 h at optimal pH and temperature. Strain ZT was able to grow on a variety of organic compounds. Most carbon sources were converted to acetate, CO2, H2, and traces of propionate and lactate. Strain ZT oxidized glutamate to acetate, CO2, NH4+, traces of propionate and H2. The doubling time on this substrate was 1-6 d. The strain fermented glutamate syntrophically in co-culture with Methanobacterium thermoautotrophicum Z-245T to the same products, but the co-culture had a fourfold higher growth rate. 16S rDNA sequence analysis revealed a relationship with Thermobrachium celere, Caloramator indicus and Caloramator proteoclasticus. The G+C content was 31.7 mol%. Based on its morphological, phylogenetic and physiological characteristics, it is proposed that strain ZT should be classified in the genus Caloramator as a new species, Caloramator coolhaasii.

Syntrophobacter fumaroxidans sp. nov., a syntrophic propionate-degrading sulfate-reducing bacterium.

A syntrophic propionate-oxidizing bacterium, strain MPOBT, was isolated from a culture enriched from anaerobic granular sludge. It oxidized propionate syntrophically in co-culture with the hydrogen- and formate-utilizing Methanospirillum hungateii, and was able to oxidize propionate and other organic compounds in pure culture with sulfate or fumarate as the electron acceptor. Additionally, it fermented fumarate. 16S rRNA sequence analysis revealed a relationship with Syntrophobacter wolinii and Syntrophobacter pfennigii. The G + C content of its DNA was 60.6 mol%, which is in the same range as that of other Syntrophobacter species. DNA-DNA hybridization studies showed less than 26% hybridization among the different genomes of Syntrophobacter species and strain MPOBT. This justifies the assignment of strain MPOBT to the genus Syntrophobacter as a new species. The name Syntrophobacter fumaroxidans is proposed; strain MPOBT (= DSM 10017T) is the type strain.

Anaerobic degradation of propionate by a mesophilic acetogenic bacterium in coculture and triculture with different methanogens.

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO(2) in cocultures with the formate- and hydrogen-utilizing methanogens Methanospirillum hungatei and Methanobacterium formicicum. Propionate oxidation did not occur in cocultures with two Methanobrevibacter strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the Methanobrevibacter strains, and organisms which are able to convert formate into H(2) plus CO(2) (Desulfovibrio strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with Methanospirillum hungatei and the aceticlastic Methanothrix soehngenii, propionate was degraded faster than in biculture with Methanospirillum hungatei, indicating that low acetate concentrations are favorable for propionate oxidation as well.

Growth of syntrophic propionate-oxidizing bacteria with fumarate in the absence of methanogenic bacteria.

Oxidation of succinate to fumarate is an energetically difficult step in the biochemical pathway of propionate oxidation by syntrophic methanogenic cultures. Therefore, the effect of fumarate on propionate oxidation by two different propionate-oxidizing cultures was investigated. When the methanogens in a newly enriched propionate-oxidizing methanogenic culture were inhibited by bromoethanesulfonate, fumarate could act as an apparent terminal electron acceptor in propionate oxidation. C-nuclear magnetic resonance experiments showed that propionate was carboxylated to succinate while fumarate was partly oxidized to acetate and partly reduced to succinate. Fumarate alone was fermented to succinate and CO(2). Bacteria growing on fumarate were enriched and obtained free of methanogens. Propionate was metabolized by these bacteria when either fumarate or Methanospirillum hungatii was added. In cocultures with Syntrophobacter wolinii, such effects were not observed upon addition of fumarate. Possible slow growth of S. wolinii on fumarate could not be demonstrated because of the presence of a Desulfovibrio strain which grew rapidly on fumarate in both the absence and presence of sulfate.

Hydrophobicities and electrophoretic mobilities of anaerobic bacterial isolates from methanogenic granular sludge.

The hydrophobicities and electrophoretic mobilities of isolates from methanogenic anaerobic granular sludge were measured and compared with those of strains from culture collections. All new isolates were highly hydrophobic, indicating that the upflow anaerobic sludge blanket reactor concept selects for hydrophobic bacteria. Methanothrix soehngenii, a methanogen often observed in methanogenic granular sludge, was highly hydrophobic and showed low electrophoretic mobility at pH 7. The role of this strain in the formation of methanogenic granular sludge is discussed.

Bacteriological composition and structure of granular sludge adapted to different substrates.

The bacteriological composition and ultrastructure of mesophilic granular methanogenic sludge from a large-scale Upflow Anaerobic Sludge Blanket reactor treating wastewater from a sugar plant and of sludge granules adapted to ethanol and propionate were studied by counting different bacterial groups and by immunocytochemical methods. Propionate-grown granular sludge consisted of two types of clusters, those of a rod-shaped bacterium immunologically related to Methanothrix soehngenii and those consisting of two different types of bacteria with a specific spatial orientation. One of these bacteria reacted with antiserum against Methanobrevibacter arboriphilus AZ, whereas the other is most likely a propionate-oxidizing bacterium immunologically unrelated to Syntrophobacter wolinii. Sludge granules obtained from the large-scale Upflow Anaerobic Sludge Blanket reactor and granules cultivated on ethanol did not show the typical spatial orientation of bacteria. Examination of the bacterial composition of the three types of granules by light and electron microscopy, the most-probable-number method, and by isolations showed that M. arboriphilus and M. soehngenii were the most abundant hydrogenotrophic and acetoclastic methanogens in propionate-grown sludge. Methanospirillum hungatei and Methanosarcina barkeri predominated in ethanol-grown granules, whereas many morphotypes of methanogens were abundant in granules from the full-scale reactor.