Population Changes of Eubacteria, Sulfate-Reducing Bacteria and Methanogenic Archaea in an Anaerobic Reactor Processing Ethanol Distillery Vinasse (original) (raw)
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Trakya University Journal of Natural Sciences, 2021
In this study, the microbial characteristics of the anaerobic reactor of a sugar industry wastewater treatment plant were analyzed using cloning, FISH (Fluoresan in situ hybridization) and metagenomic analysis. Samples were obtained from seven different ports of the reactor on the 148 th day of operation. The temperature was maintained at mesophilic conditions. The system's pH range was operated at 6.8. The cloning results showed that most of the bacterial clones belonged to uncultured members of the Bacteria domain. Many archaeal clones were related to uncultured Archaea and Methanosarcina. The FISH method was applied to determine the microbial composition of the samples, which showed that bacterial and archaeal species had nearly equal rates. Rod-shaped cells, long bacilli, coccus and long chains were detected in the samples. After metagenomic analysis, in all samples, Archaea domain members ranged between 60-36% and Bacteria domain members ranged between 58-31%. At the phylum level, in all samples, Euryarchaeota was the most dominant phylum. Proteobacteria (14.8-21.97%) and Actinobacteria (5.53-15.94%) phyla were high in rate. Furthermore, members of Spirochaeotes (0.63-4.82%) and Bacteroidetes (1.72-2.38%) were analyzed in the samples. This study revealed both bacterial and archaeal populations in the reactor of highconcentration organic sugar wastewater. These results will help in the development of more efficient anaerobic treatment systems. Özet: Çalışmada bir şeker endüstrisi atık su arıtma tesisinin anaerobik reaktörünün mikrobiyal özellikleri klonlama, FISH (Floresan in situ hibridizasyon) ve metagenomik analiz kullanılarak analiz edilmiştir. Örnekler 148. operasyon gününde reaktörün yedi farklı girişinden alınmıştır. Sıcaklık mezofilik koşullarda tutulmuştur. Sistem 6,8 pH aralığında çalışmıştır. Klonlama sonuçları, bakteri klonlarının çoğunun kültüre alınmamış Bacteria üyelerine ait olduğunu göstermiştir. Birçok arkeal klon, kültüre alınmamş Archaea ve Methanosarcina ile ilişkilidir. FISH yöntemi de örneklerin mikrobiyal kompozisyonunu belirlemek için uygulanmıştır. Elde edilen sonuçlar bakteriyel ve arkel türlerin neredeyse eşit oranlarda bulunduğunu göstermiştir. Örneklerde çubuk şeklindeki hücreler, uzun basiller, koklar ve uzun zincirler tespit edilmiştir. Metagenomik analiz sonuçları değerlendirildiğinde ise, tüm örneklerde, Archaea domaini üyelerinin %60-36 oranları arasında ve Bakteri domaini üyelerinin ise %58-31 oranları arasında bulunduğu belirlenmiştir. Filum düzeyinde, tüm örneklerde Euryarchaeota filumunun en baskın filum olduğu saptanmıştır. Proteobacteria (%14,8-21,97) ve Actinobacteria (%5,53-15,94) filumlarının da yüksek oranda olduğu görülmüştür. Ayrıca örneklerde Spirochaeotes (%0,63-4,82) ve Bacteroidetes (%1,72-2,38) üyeleri analiz edilmiştir. Bu çalışma, yapılan analizler ile yüksek konsantrasyonlu organik şeker atıksu reaktöründe hem bakteriyel hem de arkael popülasyonları ortaya çıkarmıştır. Bu sonuçlar, daha verimli anaerobik arıtma sistemlerinin geliştirilmesine yardımcı olacaktır.
Environmental Technology, 2022
Considering the scarcity of data in the literature regarding phylogenetic and metabolic composition of different inocula, especially those from thermophilic conditions, this research aimed at characterizing the microbial community and preferable metabolic pathways of an UASB reactor sludge applied to the thermophilic treatment (55°C) of sugarcane vinasse, by means of shotgun metagenomics. After its metabolic potential was depicted, it was possible to observe several genes encoding enzymes that are of great importance to anaerobic digestion processes with different wastes as substrate, especially regarding the biodegradation of carbohydrates and ligninolytic compounds, glycerolypids, volatile fatty acids and alcohols metabolism and biogas (H 2 and CH 4) production. The genera identified in higher relative abundances for Bacteria domain were Sulfirimonas (37.52 ± 1.8%), possibly related to the sludge endogenic activity due to its strong relation with a peptidoglycan lyase enzymes family, followed by Fluviicola (5.01 ± 1.0%), Defluviitoga (4.36 ± 0.2%), Coprothermobacter (4.32 ± 0.5%), Fervidobacterium (2.93 ± 0.3%), Marinospirillum (2.75 ± 0.2%), Pseudomonas (2.14 ± 0.2%) and Flavobacterium (1.78 ± 0.1%), mostly related with carbohydrates fermentations and/or H 2 production. For Archaea domain, Methanosarcina (0.61 ± 0.1%), Methanothermobacter (0.38 ± 0.0%), Methanoculleus (0.30 ± 0.1%), Thermococcus (0.03 ± 0.0%), Methanolobus (0.02 ± 1.8%), Methanobacterium (0.013 ± 0.0%), Aciduliprofundum and Pyrococcus (0.01 ± 0.0%) were the most dominant ones, being Methanosarcina the most related with methanogenesis. It was concluded that the robust inoculum description performed in this study may subside future biotechnological researches by using similar inocula (UASB sludges), focusing on the obtainment of value-added by-products by means of anaerobic digestion, such as volatile fatty acids, alcohols and biogas (H 2 and CH 4), by using several types of waste as substrate.
Process Biochemistry, 2006
Two distinct full-scale upflow anaerobic sludge blanket (UASB) reactors, namely IUASB (143 m 3 ) and TUASB (476 m 3 ), treating alcohol (raki) distillery wastewaters were investigated in terms of performance, acetoclastic methanogenic activity and archaeal composition. The TUASB reactor was inoculated with the seed sludge taken from the IUASB reactor. An average of 90% COD removal efficiency at organic loading rates (OLRs) in a range of 6-11 kg COD/m 3 day was achieved in the IUASB reactor. However, 60-80% COD removal efficiency of the TUASB reactor was achieved at OLRs ranging 2.5-8.5 kg COD/m 3 day. Both UASB reactors showed almost the same acetoclastic methanogenic activity (350 and 376 ml CH 4 /g VSS day). Distribution of archaeal populations within the two UASB reactors was investigated by denaturing gradient gel electrophoresis analysis of PCR amplified ribosomal RNA gene fragments. Both UASB reactors have shown quite similar archaeal composition (weighted similarity of 83.1%). Predominant archaeal sequences in both reactors belonged to Methanobacterium formicicum and Methanosaeta soehngenii. #
Applied and Environmental Microbiology, 1990
The bacterial population of a high-rate, anaerobic, fixed-bed loop reactor treating sulfite evaporator condensate from the pulp industry was studied over a 14-month period. This period was divided into seven cycles that included a startup at the beginning of each cycle. Some 82% of the total biomass was immobilized on and between the porous glass rings filling the reactor. The range of the total number of microorganisms in these biofilms was 2 × 10 9 to 7 × 10 9 cells per ml. Enumeration and characterization by microbiological methods and by phase-contrast, epifluorescence, and electron microscopy showed that the samples consisted mainly of the following methanogens: a Methanobacterium sp., a Methanosarcina sp., a Methanobrevibacter sp., and a Methanothrix sp., as well as furfural-degrading sulfate-reducing bacteria resembling Desulfovibrio furfuralis. Viable counts of hydrogenotrophic methanogens were relatively stable (mostly within the range of 3.2 × 10 8 to 7.5 × 10 8 cells per ...
Applied Microbiology and Biotechnology, 2012
The microbial communities (Bacteria and Archaea) established in an anaerobic fluidized bed reactor used to treat synthetic vinasse (betaine, glucose, acetate, propionate, and butyrate) were characterized by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. This study was focused on the competitive and syntrophic interactions between the different microbial groups at varying influent substrate to sulfate ratios of 8, 4, and 2 and anaerobic or micro-aerobic conditions. Acetogens detected along the anaerobic phases at substrate to sulfate ratios of 8 and 4 seemed to be mainly involved in the fermentation of glucose and betaine, but they were substituted by other sugar or betaine degraders after oxygen application. Typical fatty acid degraders that grow in syntrophy with methanogens were not detected during the entire reactor run. Likely, sugar and betaine degraders outnumbered them in the DGGE analysis. The detected sulfate-reducing bacteria (SRB) belonged to the hydrogen-utilizing Desulfovibrio. The introduction of oxygen led to the formation of elemental sulfur (S 0 ) and probably other sulfur compounds by sulfide-oxidizing bacteria (γ-Proteobacteria). It is likely that the sulfur intermediates produced from sulfide oxidation were used by SRB and other microorganisms as electron acceptors, as was supported by the detection of the sulfur respiring Wolinella succinogenes. Within the Archaea population, members of Methanomethylovorans and Methanosaeta were detected throughout the entire reactor operation. Hydrogenotrophic methanogens mainly belonging to the genus Methanobacterium were detected at the highest substrate to sulfate ratio but rapidly disappeared by increasing the sulfate concentration.
Environmental science and pollution research international, 2017
Performance and microbial community composition were evaluated in a two-phase anaerobic and aerobic system treating sulfate-rich cellulosic ethanol wastewater (CEW). The system was operated at five different chemical oxygen demand (COD)/SO4(2-) ratios (63.8, 26.3, 17.8, 13.7, and 10.7). Stable performance was obtained for total COD removal efficiency (94.5%), sulfate removal (89.3%), and methane production rate (11.5 L/day) at an organic loading rate of 32.4 kg COD/(m(3)·day). The acidogenic reactor made a positive contribution to net VFAs production (2318.1 mg/L) and sulfate removal (60.9%). Acidogenic bacteria (Megasphaera, Parabacteroides, unclassified Ruminococcaceae spp., and Prevotella) and sulfate-reducing bacteria (Butyrivibrio, Megasphaera) were rich in the acidogenic reactor. In the methanogenic reactor, high diversity of microorganisms corresponded with a COD removal contribution of 83.2%. Moreover, methanogens (Methanosaeta) were predominant, suggesting that these organi...
Environmental science and pollution research international, 2015
This study investigated the microbial diversity established in a combined system composed of a continuous stirred tank reactor (CSTR), expanded granular sludge bed (EGSB) reactor, and sequencing batch reactor (SBR) for treatment of cellulosic ethanol production wastewater. Excellent wastewater treatment performance was obtained in the combined system, which showed a high chemical oxygen demand removal efficiency of 95.8 % and completely eliminated most complex organics revealed by gas chromatography-mass spectrometry (GC-MS). Denaturing gradient gel electrophoresis (DGGE) analysis revealed differences in the microbial community structures of the three reactors. Further identification of the microbial populations suggested that the presence of Lactobacillus and Prevotella in CSTR played an active role in the production of volatile fatty acids (VFAs). The most diverse microorganisms with analogous distribution patterns of different layers were observed in the EGSB reactor, and bacteri...
Anaerobe, 2008
In this paper, the microbial characteristics of the granular sludge in the presence of oxygen (3.070.7 mg O 2 l À1 ) were analyzed using molecular biology techniques. The granules were provided by an upflow anaerobic sludge blanket (UASB) operated over 469 days and fed with synthetic substrate. Ethanol and sulfate were added to obtain different COD/SO 4 2À ratios (3.0, 2.0, and 1.6). The results of fluorescent in situ hybridization (FISH) analyses showed that archaeal cells, detected by the ARC915 probe, accounted for 77%, 84%, and 75% in the COD/SO 4 2À ratios (3.0, 2.0, and 1.6, respectively).
Applied and Environmental Microbiology, 1991
Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35°C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-C02. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens. Methanogenic granules are self-immobilized consortia of methanogens, syntrophic acetogens, and hydrolytic-fermen-tative bacteria that convert soluble organic matter to methane and CO2. The published work on the granule formation, microbial composition, substrate conversion potentials, and microbial structures of granules has focused on methanogens and syntrophic acetogens (7-11, 15, 23, 32, 39). This is likely due to the observation that sulfate-reducing bacteria (SRB) are present at much lower levels than syntrophic acetogens in granules treating sugar wastewater (10). Considerable information as to the methanogenic degradation of acetate, formate, and H2-CO2 in granules has been reported (7, 8, 11). Little work, however, has been performed to evaluate the conversion of other common substrates such as ethanol, propionate, and butyrate. SRB are quite diverse in terms of metabolic activities, morphotypes, trophic properties, and substrate affinities. In the presence of sulfate, acetate can be oxidized to CO2 by some pure SRB cultures; propionate, butyrate, and other volatile fatty acids (VFAs) can be oxidized completely to * Corresponding author. CO2 or converted to acetate or acetate plus propionate (in the case of odd long-chain acids with five or more carbon atoms); branched fatty acids such as isobutyrate, isovaler-ate, and 2-methylbutyrate can also be oxidized completely to CO2 or incompletely to acetate (35, 37). Hydrogen and formate can be utilized by many SRB as electron donors for sulfate reduction (35). Acetate and methanol are degraded via sulfate reduction by a coculture consisting of Desulfo-vibrio vulgaris and Methanosarcina barkeri (22). Methanol can be also degraded to CO2 via sulfate reduction by a coculture consisting of Desulfovibrio vulgaris and various homoacetogens (6, 12). In the absence of sulfate, certain SRB such as Desulfovibrio spp. may grow together with H2-utilizing methanogens to convert ethanol or lactate to acetate syntrophically (18, 34). The existence of syntrophic associations between H2-producing SRB and H2-consuming methanogens in lake sediments was suggested (5). No mention of syntrophic catabolism of VFAs by SRB in granular systems has been reported. VFAs such as propionate and butyrate are thought to be converted only by obligate syntrophic acetogens in concert with H2-utilizing methano-gens (3, 19, 20, 28, 35). In this study, methanogenic granules grown on brewery 3438