Start up of biological sequencing batch reactor (SBR) and short‐term biomass acclimation for polyhydroxyalkanoates production

BACKGROUND: The adaptation/selection of mixed microbial cultures under feast/famine conditions is an essential step for polyhydroxyalkanoates (PHA) production. This study investigated the short‐term adaptation of a mixed microbial culture (activated sludge) during the start up of a sequencing batch reactor (SBR). RESULTS: Four different SBR runs were performed starting from different inocula and operated at the same organic load rate (8.5 gCOD L^?1 d^?1) and hydraulic retention time (1 day). At 3–7 days from SBR start up, the selected biomass was able to store PHA at comparable rate and yield with those obtained after long‐term acclimation. Independently from the time passed, a short feast phase was the key parameter to obtain PHA storage at high rate and yield in the following accumulation stage (244 mgCOD g^?1CODnonPolym h^?1 for specific storage rate and 48% COD COD^?1 as PHA content in the biomass). The DGGE profiles showed that the good storage performance and the structure of the microbial community were not fully correlated. CONCLUSIONS: The results suggest a new strategy for operating the PHA accumulation stage directly in the SBR, after very short biomass adaptation, instead of using two separate reactors for biomass enrichment and PHA accumulation, respectively. © 2012 Society of Chemical Industry     

Exploiting olive oil mill effluents as a renewable resource for production of biodegradable polymers through a combined anaerobic–aerobic process

BACKGROUND: The performance of a three‐stage process for polyhydroxyalkanoate (PHA) bioproduction from olive oil mill effluents (OME) has been investigated. In the first anaerobic stage OME were fermented in a packed bed biofilm reactor into volatile fatty acids (VFAs). This VFA‐rich effluent was fed to the second stage, operated in an aerobic sequencing batch reactor (SBR), to enrich mixed cultures able to store PHAs. Finally, the storage response of the selected consortia was exploited in the third aerobic stage, operated in batch conditions. RESULTS: The anaerobic stage increased the VFA percentage in the OME from 18% to ～32% of the overall chemical oxygen demand (COD). A biomass with high storage response was successfully enriched in the SBR fed with the fermented OME at an organic load rate of 8.5 gCOD L^?1 d^?1, with maximum storage rate and yield (146 mgCOD gCOD^?1 h^?1 and 0.36 COD COD^?1, respectively) very similar to those obtained with a synthetic VFA mixture. By means of denaturing gradient gel electrophoresis (DGGE) analysis, different bacterial strains were identified during the two SBR runs: Lampropedia hyalina and Candidatus Meganema perideroedes, with the synthetic feed or the fermented OMEs, respectively. In the third stage, operated at increasing loads, the maximum concentration of the PHA produced increased linearly with the substrate fed. Moreover, about half of the stored PHAs were produced from substrates other than VFAs, mostly alcohols. CONCLUSION: The results obtained indicate that the process is effective for simultaneous treatment of OME and their valorization as a renewable resource for PHA production. Copyright © 2009 Society of Chemical Industry     

Bioconversion of residual soybean oil into polyhydroxyalkanoates

The aim of this study is to evaluate the bioconversion of residual soybean oil (RSO) into polyhydroxyalkanoates (PHAs) by selecting microorganism and fermentation condition in order to increase PHAs production. PHAs production by Cupriavidus necator IPT 026 using glucose (PHA 1) and RSO (PHA 2) as substrate is 1.15 ± 0.21 and 2.84 ± 0.04 g L^?1, respectively. FTIR spectra of PHAs were similar to data reported in literature. PHAs presented low crystallinity (PHA 1: 42.69%; PHA 2: 46.44%), high thermal stability (PHA 1: 271.78 °C; PHA 2: 272.52 °C), and low M_W (PHA 1: 140.69 kDa; PHA 2: 254.54 kDa). PHAs produced by RSO are potential candidates for industrial applications, especially ones that demand higher temperatures. This is the first study on the production and characterization of PHAs obtained by C. necator IPT 026 in culture with RSO. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46255.     

In vitro production of polyhydroxyalkanoates: achievements and applications

The mechanisms of polyhydroxyalkanoate (PHA) production have been studied for over half a century. However, despite numerous improvements in the control of monomer composition, genetically‐engineered host organisms, fermentation strategies and polymer recovery processes they remain uncompetitive compared with petrochemical plastics. Recently, interest has developed in the enzyme‐catalysed production of PHAs in vitro. This has allowed the study of enzyme kinetics and properties, and represents another strategy for the economic production of PHAs on the industrial scale. It also presents an opportunity to coat other materials in thin films of PHA so as to modify the surface properties. In vitro production offers advantages over in vivo methods as it enables greater control over monomer composition and molecular weight, does not require a biomass‐accumulation phase, simplifies downstream processing and can utilise a wider range of monomeric subunits. Copyright © 2009 Society of Chemical Industry     

Modeling and simulation of the formation and utilization of microbial products in aerobic granular sludge

A mathematical model is established to simulate the formation of extracellular polymeric substances (EPS), soluble microbial products (SMP), and internal storage products (X_STO) in aerobic granular sludge. The sensitivity of these microbial products concentrations toward the key model parameters is analyzed. Independent experiments are conducted to find required parameter values and to test its predictive ability. The model is evaluated by using one‐cycle operating experimental results of a lab‐scale aerobic granule‐based sequencing batch reactor (SBR) and batch experimental results. Results show that the model is able to describe the microbial product dynamics in aerobic granules and provide further insights into a granule‐based SBR. The effect of the initial substrate and biomass concentrations on the formation of microbial products in aerobic granular sludge can therefore be analyzed by model simulation. A higher substrate concentration results in a greater concentration of EPS, SMP, and X_STO. An accumulation of biomass in the bioreactor leads to an increased production rate of EPS, SMP, and X_STO. © 2009 American Institute of Chemical Engineers AIChE J, 2010     

Partial nitrification of sludge reject water using suspended and granular biomass

BACKGROUND: Partial nitrification–Anammox is a combined promising advanced biological process for the removal of nitrogen from wastewater, which allows important savings in energy consumption, sludge production, and organic carbon. Granular biomass appears to be an interesting alternative to conventional activated sludge, mainly because of its better settling properties. This study deals with the experimental results of a comparison between a conventional and a granular sequencing batch reactor (SBR) for the partial nitrification of reject water. RESULTS: After some days of operation, 30 days in the conventional SBR (system A) and 100 days in the granular SBR (system B), partial nitrification was achieved. Granular sludge showed much better settling properties than suspended biomass, with values of sludge volumetric index (SVI₁₀) of 130 mL g^?1 in system A and 38 mL g^?1 in system B. Consequently, the solids concentration within the granular reactor was three times higher than for the conventional system while the concentration of solids in the effluent was 10 times higher in the conventional SBR. Morphology, microstructure and microbial populations in both systems were also studied. CONCLUSION: A partial nitrification process was successfully achieved in both systems, obtaining an effluent with a NO₂^?‐N/NH₄⁺‐N ratio near 1, suitable for a following Anammox process. Granular biomass, mostly formed by round particles, showed better settling properties, leading to better sludge–effluent separation as well as higher biomass retention in the reactor. The granulation process does not affect bacterial populations, since they were the same in both systems. Copyright © 2011 Society of Chemical Industry     

Sequential batch reactor for eucalypt kraft pulp effluent treatment with Trametes versicolor

BACKGROUND: A sequential batch reactor (SBR) was used for eucalypt kraft pulp effluent treatment with Trametes versicolor. A 2³ full factorial design and response surface methodology were applied to optimise the batch fermentation conditions. Effluent concentration, culture medium and inoculum age were the factors selected for this study in order to optimise the reduction of chemical oxygen demand (COD). RESULTS: The presence of Trametes Defined Medium (TDM) in the fermentation was required to obtain a significant COD reduction. Experiments in the batch reactor confirmed, in general, the predicted results of optimisation developed from Erlenmeyer batch assays. The T. versicolor culture remained active during 42 days of study in the SBR, providing approximately 80% of COD reduction. CONCLUSION: Trametes versicolor may be considered as very promising for the biological treatment of effluents from kraft pulp mills in an SBR system instead of the activated sludge mixed cultures traditionally used. Copyright © 2008 Society of Chemical Industry     

Simultaneous organic carbon and nitrogen removal in an SBR controlled at low dissolved oxygen concentration

Simultaneous organic carbon and nitrogen removal was studied in a sequencing batch reactor (SBR) fed with synthetic municipal wastewater and controlled at a low dissolved oxygen (DO) level (0.8 mg dm^?3). Experimental results over a long time (120 days) showed that the reactor achieved high treatment capacities (organic and nitrogen loading rates reached as high as 2.4 kg COD m^?3 d^?1 and 0.24 kg NH₃‐N m³ d^?1) and efficiencies (COD, NH₃‐N and total nitrogen removal efficiencies were 95%, 99% and 75%). No filamentous bacteria were found in the sludge even though the reactor had been seeded with filamentous bulking sludge. Instead, granular sludge, which possessed high activity and good settleability, was formed. Furthermore, the sludge production rate under low DO was less than that under high DO. Significant benefits, such as low investment and less operating cost, will be obtained from the new process. © 2001 Society of Chemical Industry     

Comparative analysis of different properties of polyhydroxyalkanoates isolated from two different bacterial strains: Alkaliphilus oremlandii OhILAs and recombinant Escherichia coli XL1B

We synthesized poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) [P(3‐HB‐co‐3‐HV)] copolymer having different contents of 3‐hydroxyvalerate (3‐HV) units (16.04, 16.3, 24.95, 25.62, and 16.52 mol % 3‐HV) with different yields of polyhydroxyalkanoates (PHAs) by feeding with different cooking oils and with Alkaliphilus oremlandii OhILAs strain. The PHA production efficiency of the Alkaiphilus strain was compared with that of the control strain, Bacillus cereus. The synthesis of each PHA biopolymer was performed with different toxic spent oils as the sole carbon source in an oil‐in‐water‐based microemulsion medium. We observed that the productivity of the poly(3‐hydroxybutyrate) [P(3‐HB)] copolymer from the Alkaliphilus strain was higher than those of the PHAs isolated from B. cereus and the Escherichia coli XL1B strain. The synthesized PHA copolymers were characterized by ¹H‐NMR and Fourier transform infrared (FTIR) spectroscopy. In the ¹H‐NMR spectra, a doublet resonance peak at 1.253 ppm of the/ methyl protons of the 3‐hydroxybutyrate (3‐HB) side group and one at 0.894 ppm due to the methyl protons of the 3‐HV side group indicated the presence of 3‐HB and 3‐HV units in the copolymer. The chemical shift values at 1.25 and 2.2 ppm, due to the resonance absorption peaks of the methyl protons and methylene protons, confirmed the synthesis of the P(3‐HB) homopolymer. From the FTIR spectra, a strong C?O stretching frequency in the range of 1745–1727 cm^?1, together with strong C? O stretching bands near 1200 cm^?1 and a strong band near 3400 cm^?1, confirmed the synthesis of P(3‐HB‐co‐3‐HV) and P(3‐HB). Thus, waste cooking oil as a substrate provided an alternate route for the formation of P(3‐HB‐co‐3‐HV) and P(3‐HB) by Alkaliphilus and E. coli strains, respectively. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41080.     

Effect of Media Components and Growth Conditions for Improved Linoleic Acid Production by Beauveria Species

Beauveria species are well-known insect pathogenic fungi, and Beauveria bassiana is used as a biopesticide against various pests in agriculture. However, the Beauveria species has not been reported as producers of microbial oils. In this study, Beauveria spp. MTCC 5184 was used to produce microbial oil with high linoleic acid (LA) content. Ten experiments were performed to evaluate the effects of several media parameters, such as carbon and nitrogen sources, pH, various concentrations of carbon and nitrogen, growth duration, and oleic acid (OLA) supplementation for maximum LA and dry biomass production by the fungus. Several of these parameters had a significant impact on the production of LA, as well as dry biomass. The glucose yeast extract (GYE) medium supplemented with 1.5% (w/v) peptone yielded maximum LA (0.32 ± 0.01 g L⁻¹) and biomass (5.51 ± 0.26 g L⁻¹). However, through the addition of 1.0% (w/v) OLA, the precursor of LA, LA production was enhanced 12-fold (1.24 ± 0.03 g L⁻¹), and the biomass production increased by 5-fold (11.05 ± 0.46 g L⁻¹) in comparison to those in the basal (GYE) medium. Using lactose as the sole carbon source produced the lowest LA (0.05 ± 0.00 g L⁻¹) and biomass (1.04 ± 0.10 g L⁻¹). The results of this study will be useful for the commercial exploitation of this fungus for the production of LA-rich microbial oil for use in the production of lubricants, greases, paints, cosmetics, etc.     

Comparative study on the effect of various pretreatment methods on the enrichment of hydrogen producing bacteria in anaerobic granulated sludge from brewery wastewater

Five pretreatment methods, namely chemical, acid, heat-shock, freezing and thawing, and base, were evaluated for the enrichment of hydrogen-producing bacteria in anaerobic granulated sludge, which will be subsequently used as seed in biological hydrogen production. All the pretreatments showed positive effects towards improving hydrogen (H₂) generation by the microbial population with higher hydrogen production yield and COD removal efficiency as compared to control. The granulated sludge pretreated by heat-shock showed maximum accumulated H₂ (19.48mLg^?1-COD), COD removal efficiency (62%), and biomass concentration (22.5 gL^?1).     

A three‐phase fluidized bed reactor in the combined anaerobic/aerobic treatment of wastewater

Aerobic degradation or polishing is an essential step in the combined anaerobic/aerobic treatment of wastewater. In this study, a type of porous glass beads was used for immobilization of microbial cells in a three‐phase aerobic fluidized bed reactor (AFBR) with an external liquid circulation. The effects of superficial gas and liquid velocities on bed expansion, solid and gas hold‐ups and specific oxygen mass transfer rate, k_La, were investigated. A tracer study showed that the mixing and flow pattern in the 8 dm³ reactor could be simulated by a non‐ideal model of two continuous stirred tank reactors (CSTRs) in series. By treating an effluent from an upflow anaerobic sludge blanket (UASB) digester, the distribution of suspended and immobilized biomass in the reactor as well as the kinetics of COD removal were determined. The specific oxygen mass transfer rate, k_La, at a superficial gas velocity of 0.7 cm s⁻¹ dropped by about 30% from 32 h⁻¹ in tap water to 22 h⁻¹ after a carrier load of 15% (v/v) was added. The measured k_La further dropped by about 20% to 18 h⁻¹ in the wastewater, a typical value of the bubbling fermenters with no stirring. Compared with the aerobic heterotrophs under optimum growth conditions, the microbes in this reactor which was fed with anaerobic effluent plus biomass behaved like oligotrophs and showed slow specific COD removal rates. This might be attributed to the presence of a significant amount of obligate anaerobes and facultative organisms in the aerobic reactor. This was confirmed by a relatively low intrinsic oxygen uptake rate of the microbial population in the reactor, 94 mg O₂ dm⁻³ h⁻¹ or 19 mg O₂g VS⁻¹ h⁻¹. © 1999 Society of Chemical Industry     

Modelling a sequencing batch reactor to treat the supernatant from anaerobic digestion of the organic fraction of municipal solid waste

In this study, a lab‐scale sequencing batch reactor (SBR) has been tested to remove chemical oxygen demand (COD) and NH₄⁺‐N from the supernatant of anaerobic digestion of the organic fraction of municipal solid waste. This supernatant was characterized by a high ammonium concentration (1.1 g NH₄⁺‐N L^?1) and an important content of slowly biodegradable and/or recalcitrant COD (4.8 g total COD L^?1). Optimum SBR operating sequence was reached when working with 3 cycles per day, 30 °C, SRT 12 days and HRT 3 days. During the time sequence, two aerobic/anoxic steps were performed to avoid alkalinity restrictions. Oxygen supply and working pH range were controlled to promote the nitrification over nitrite. Under steady state conditions, COD and nitrogen removal efficiencies of more than 65% and 98%, respectively, were achieved. A closed intermittent‐flow respirometer was used to characterize and model the SBR performance. The activated sludge model ASM1 was modified to describe the biological nitrogen removal over nitrite, including the inhibition of nitrification by unionized ammonia and nitrous acid concentrations, the pH dependency of both autotrophic and heterotrophic biomass, pH calculation and the oxygen supply and stripping of CO₂ and NH₃. Once calibrated by respirometry, the proposed model showed very good agreement between experimental and simulated data. Copyright © 2007 Society of Chemical Industry     

Aerobic granules for low‐strength wastewater treatment: formation,structure, and microbial community

BACKGROUND: To validate the possibility of aerobic granulation at a lower organic loading rate (OLR) than 2 kg COD m^?3 day^?1 (GS 1) in a sequencing batch reactor (SBR), the formation, structure, and microbial community of granular sludge (GS) were investigated. RESULTS: The overall experimental process involved the following stages: acclimation, granulation, maturation, and stabilization. The optical microscopic showed the structural changes from fluffy activated sludge (AS) to GS and scanning electron microscope (SEM) examination revealed that GS 1 was irregular filamentous aggregates composed mainly of various filamentous species, while the aerobic granules cultivated at OLR 1.68–4.20 kg COD m^?3 day^?1 (GS 2) was mycelial pellets consisting of fungi and filamentous microorganisms. A Biolog Ecoplate analysis indicated that significant differences existed between the microbial community structure and the substrate's utilization of AS and different GS samples. CONCLUSION: GS 1 was achieved and different from GS 2 in the formation, structure, and microbial community. Aerobic granulation with low strength wastewater is of importance for the full‐scale application of this technology. Copyright © 2009 Society of Chemical Industry     

Production of polyhydroxyalkanoates: the future green materials of choice

Polyhydroxyalkanoates (PHAs) have recently been the focus of attention as a biodegradable and biocompatible substitute for conventional non degradable plastics. The cost of large‐scale production of these polymers has inhibited its widespread use. Thus, economical, large‐scale production of PHAs is currently being studied intensively. Various bacterial strains, either wild‐type or recombinant have been utilized with a wide spectrum of utilizable carbon sources. New fermentation strategies have been developed for the efficient production of PHAs at high concentration and productivity. With the current advances, PHAs can now be produced to a concentration of 80 g L^?1 with productivities greater than 4 g PHA L^?1 h^?1. These advances will further lower the production cost of PHAs and allow this family of polymers to become a leading biodegradable polymer in the near future. This review describes the properties of PHAs, their uses, the various attempts towards the production of PHAs, focusing on the utilization of cheap substrates and the development of different fermentation strategies for the production of these polymers, an essential step forward towards their widespread use. Copyright © 2010 Society of Chemical Industry     

The effect of COD/N ratio on process performance and membrane fouling in a submerged bioreactor

Influent chemical oxygen demand/nitrogen (COD/N) ratio is used to control fouling in membrane bioreactor (MBR) systems. However, COD/N also affects the physicochemical and biological properties of MBR biomass. The current study examined the relationship between COD/N ratio in feed wastewater and extracellular polymeric substances (EPS) production in MBRs. Two identical submerged MBRs with different COD/N ratios of 10:1 and 5:1 were operated in parallel. The cation concentration and floc-size of the sludge were measured. The composition and characteristics of bound EPS and soluble microbial products (SMP) under each COD/N ratio were also examined. Batch tests were conducted in 1000 mL bottles to study the process of the release of foulants from the sludge when 1 g of (NH₄⁺-N)/L was added. Results showed that the influent COD/N ratio could change the physicochemical properties of EPS and SMP. Moreover, excessive NH₄⁺ in the supernatant could facilitate the role of NH₄⁺ as a monovalent cation, the replacement of the polyvalent cation in bound EPS, and even the extraction of EPS components from the surface of the sludge to form new SMP.     

Semibatch and continuous fructooligosaccharides production by Aspergillus sp. N74 in a mechanically agitated airlift reactor

BACKGROUND: Fructooligosaccharides are important sweeteners produced by sucrose biotransformation. Although fructooligosccharides production has been reported widely, most studies have been carried out at laboratory level. This study evaluates semibatch and continuous fructooligosaccharides production by Aspergillus sp. N74 at bench scale in a mechanically agitated airlift. RESULTS: Sucrose biotransformation to fructooligosaccharides was carried out with biomass harvested after 24 or 48 h of culture. For 6.21 ± 0.33 or 9.66 ± 0.62 g biomass dry weight L⁻¹, the highest FOS yields were obtained at batch operating 62.1 and 66.4% after 26 or 6 h of reaction, respectively. Reduction in fructooligosaccharides yield was observed for both biomass concentrations at semibatch operating, while a comparable yield was obtained during continuous operating (62.1% for 6.21 ± 0.33 g L⁻¹ and a dilution rate 0.016 s⁻¹, and 62.8% for 9.66 ± 0.62 g L⁻¹ and a dilution rate 0.032 s⁻¹). Nevertheless, 1‐kestose formation was favored with biomass harvested after 24 h under any operating mode. CONCLUSION: Biomass concentration, reaction time and operating mode have a notable effect on fructooligosaccharides yield and composition. 1‐kestose, the most valuable fructooligosaccharide, was obtained in greatest proportion at a biomass concentration 6.21 ± 0.33 g L⁻¹. Under the different operating modes, Aspergillus sp. N74 mycelia and the reactor described are presented as a feasible alternative for scaling up fructooligosaccharides production. Copyright © 2008 Society of Chemical Industry     

The operational strategy of intermittent cycle extended aeration system in treating sewage

An Intermittent Cycle Extended Aeration System (ICEAS) offers advantages for treating sewage; such as easy operation, process flexibility, and low capital cost. A laboratory‐scale study was made with synthetic‐domestic wastewater (COD = 300 mg dm⁻³; BOD = 210 mg dm⁻³) to investigate appropriate conditions for reduced operating cost. The results from this study indicated that the maximum hydraulic loading and organic loading were 3.5 m³ m⁻³ d⁻¹ and 0.735 kg BOD m⁻³d⁻¹ respectively. The BOD and COD of effluent were 15.5 mg dm⁻³ and 29.6 mg dm⁻³ for the cycle time and aeration time of 3.4 h and 2.65 h. It was not necessary to supply external artificial substrates in the reactor to deal with low wastewater flow that caused the starvation of sludge. Specific oxygen uptake rate (SOUR) was used as the index of microbial activity. The study indicated that the microbial activity could be restored (SOUR = 20.5 mg g⁻¹ MLVSS h⁻¹) after 5–6 days of cultivation when the sludge was deprived of substrate for 17 days. © 1999 Society of Chemical Industry     

Screening and selection of microfungi for microbial biomass protein production and water reclamation from starch processing wastewater

Thirty strains of microfungi and amylolytic yeasts were screened for production of microbial biomass protein (MBP) and water reclamation from starch processing wastewater (SPW). Three species and six strains of microfungi Aspergillus oryzae, Rhizopus oligosporus and Rhizopus arrhizus showing high enzymatic activities on SWP were selected under non‐aseptic growth conditions. In 20 h submerged cultivation the selected strains had a high capacity to enzymatically hydrolyse more than 93% of the starch and produce 4.3–5.6 g dm⁻³ of dry biomass at a specific biomass growth rate from 0.05 to 0.12 h ⁻². The fungal biomass contained crude protein ranging from 37.5 to 49.8% of dry biomass. The pellet and flocculated biomass products were easily harvested by simple filtration or sedimentation. After these processes, 76–88% of total organic carbon (TOC), 85%–92% of chemical oxygen demand (COD) and 95% suspended solids in SPW were removed, and the treated water was reusable for farm irrigation. Typical pretreatment processes including hydrolysis, sterilisation and nutrient supplementation were unnecessary. © 1999 Society of Chemical Industry     

Comparative evaluation of full‐scale UASB reactors treating alcohol distillery wastewaters in terms of performance and methanogenic activity

In this study, two full‐scale upflow anaerobic sludge blanket (UASB) reactors, namely TUASB and CUASB, at the wastewater treatment plants of the Tekirdaǧ Alcohol (Raki) and Canakkale Alcohol (Cognac) distilleries were investigated in terms of performance, acetoclastic methanogenic capacity and microbial composition. The results were compared with a previously studied other UASB reactor (IUASB) at the wastewater treatment plant of the Istanbul Alcohol (Raki) Distillery from which the two reactors (TUASB and CUASB) were seeded. The IUASB reactor performed well achieving COD removal efficiencies of no lower than 85% at organic logding rates (OLRs) in the range of 6–11 kg COD m⁻³ day⁻¹ between 1996 and 2001. During the last one year of operation, between 2000 and 2001, performance of the CUASB reactor in terms of COD removal efficiency was 70–80% at OLRs in a range of 1–4.5 kg COD m⁻³ day⁻¹ whereas it was 60–80% at OLRs in a range of 2.5–8.5 kg COD m⁻³ day⁻¹ in the TUASB reactor. At the end of year 2000, specific methanogenic activity (SMA) tests were carried out to determine potential loading capacity and optimum operating conditions of the IUASB, CUASB and TUASB reactors. The potential methane production (PMP) rates of the CUASB, IUASB and TUASB reactors were measured as 230 cm³ CH₄ gVSS⁻¹ day⁻¹, 350 cm³ CH₄ gVSS⁻¹ day⁻¹ and 376 cm³ CH₄ gVSS⁻¹ day⁻¹ respectively. When the PMP rates were compared with actual methane production (AMP) rates obtained from the three UASB reactors, AMP/PMP ratios were evaluated to be 0.18, 0.12 and 0.13 for CUASB, TUASB and IUASB reactors respectively. This showed that the CUASB, TUASB and IUASB reactors were using only 18%, 12% and 13% of their potential acetoclastic methanogenic capacity respectively. These results can be interpreted that the three UASB reactors were underloaded compared with their potential acetoclastic methanogenic capacities. It was, therefore, recommended that the three UASB reactors should be loaded at higher organic loading rates or sludge withdrawn in order to maintain an AMP/PMP ratio of 0.6–0.7, which can ensure desired reactor performance with safer operation. Results of epifluoresence microscopic examinations showed that the percentage of total autofluorescent methanogens was approximately 30% of the total population in sludges from the TUASB and IUASB reactors whereas it was 20% in sludge from the CUASB reactor. The two UASB reactors treating raki distillery wastewaters contained sludges having a higher percentage of autofluorescent methanogenic population and higher acetoclastic methanogenic activity. Copyright © 2004 Society of Chemical Industry