Biodegradability and toxicity of styrene in the anaerobic digestion process

Start-up and operation of an Upflow Anaerobic Sludge Blanket (UASB) reactor fed with an industrial effluent from a polymer synthesis plant containing 6 mg styrene 1ˉ¹ was unstable. In batch assays with 200 mg styrene 1ˉ¹, 74% of styrene was degraded at a rate of 7 ml methane gˉ¹ volatile suspended solids.day, without a lag phase. The toxicity limit (IC50) of styrene was 1.4 mM for the acetoclastic activity, 0.45 and 1.6 mM for the methanogenic activity in the presence of 30 mM of propionate and ethanol respectively. Instability of UASB operation was attributed to other compounds such as acrylates or detergents present in the industrial effluent.Project ALFA - ALR/B-73011/94.04-4.0181.9

Análisis de imagen para la cuantificación de cambios morfológicos en biomasa granular, inducidos por la presencia de nitrato en reactores EGSB

Cuatro reactores EGSB (R1-R4) fueron operados a velocidades de carga orgánica de 10 kgCOD/(m³.d), tiempos de retención hidráulicos de 3.6 h, velocidades superficiales de 10 m/h y diferentes concentraciones de nitrato a la entrada, 0, 20, 60 y 100 mgN-NO3-/L en R1, R2, R3 and R4, respectivamente. Después de 20 días de operación continua, la biomasa granular fue caracterizada en términos de: (i) morfología por análisis de imagen cuantitativo; (ii) actividad metanogénica específica en presencia de acetato, butirato, propionato, etanol y una mezcla gaseosa de H2CO2; velocidad de sedimentación. El parámetro LfA, definido como el cuociente entre la longitud total de filamentos y el área total proyectada, fue determinado y se observó que es suficientemente sensible para la cuantificación de diferencias morfológicas inducidas por la presencia de nitrato. Aunque el crecimiento de estructuras filamentosas en la superficie de los gránulos fue evidente, no hubo una disminución notable en la velocidad de sedimentación de los gránulos, probablemente porque durante el período de operación los gránulos crecieron en tamaño. En el reactor R4 se observó un mayor washout que en R1, R2 y R3.Fundação para a Ciência e Tecnologia (FCT

Recovery of acetoclastic activity in anaerobic granular sludge, monitored by methanogenic activity measurements and image analysis

The specific acetoclastic activity (SAA) of anaerobic granular sludge was recovered in a lab scale EGSB reactor. The inoculum presented a very low acetoclastic activity (0.81+/-2.48mg COD-CH4/gVSS.day). Ethanol was fed at a COD concentration of 1.5g/L, as sole organic carbon source to promote the biogas production through the hydrogenophilic pathway. During the operation period the specific methanogenic activity (SMA)1 in the presence of acetate, propionate, butyrate, ethanol and H2/CO2, and the biomass morphology were quantified. The SAA steadily increased as expected by the reactor performance, achieving a value of 183+/-13mg COD-CH4/gVSS.day at day 108. From the SMA results it was suggested that, until day 62, the initial methane production rate was dominated by the hydrogenotrophic population and afterwards, the developed acetoclastic activity started to play a role in the initial methane production from ethanol. Until day 62 the ratio between the filaments length and aggregates projected area (LfA)2 increased due to the breakdown of granules inside the reactor, after that decreased and granules development was simultaneous with the increase of the SAA. Percentage of aggregates in size range 0.1 to 1mm (equivalent diameter) increased until day 40 due to granules fragmentation. When the system achieved a steady state, the aggregates size increased

Image analysis to quantify morphological changes in granular sludge induced by nitrate in EGSB reactors

Four EGSB reactors (R1 to R4) were operated at an organic loading rate of 10 kgCOD/(m³.d), hydraulic retention time of 3.6 h, upflow velocity of 10 m/h and different influent nitrate concentrations (0, 20, 60 and 100 mgN-NO_3-/L for R1, R2, R3 and R4, respectively. After about 20 days of operation, the granular sludge was characterized for: (i) morphology by quantitative image analysis; (ii) specific methanogenic activity in the presence of acetate, propionate, butyrate, ethanol and H_2/CO_2; (iii) settling velocity. LfA parameter, defined as the ratio of total filaments length to total projected area, was determined and was sensitive to quantify the different surface morphology, induced by nitrate to the granular sludge. Although the growth of filamentous structures was evident, no significant decrease on the settling velocity was observed, most likely because granules grew during the trial period. A higher washout occurred in R4, as compared to R1, R2 and R3.Grant SFRH/BD/3187/2000.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI/BD/20325/99

Quantitative image analysis as a diagnostic tool for identifying structural changes during a revival process of anaerobic granular sludge

Due to unspecified operational problems, the specific acetoclastic activity (SAA) of the anaerobic granular sludge present in an industrial UASB reactor was considerably damaged (from 250 to less than 10mL CH4@STP/gVSS.d), significantly reducing the biogas production of that industrial unit. The hydrogenotrophic methanogenic activity exhibited a value of 600mL CH4@STP/gVSS.d, the settling velocity was 31.479.8 m/h, the average equivalent diameter was 0.9270.43mm, and about 70% of the VSS were structured in aggregates larger than 1mm. In order to study the recovery of the SAA, this sludge was collected and inoculated in a lab-scale expanded granular sludge blanket (EGSB) reactor. Ethanol was fed as the sole carbon source during a trial period of 106 days. Process monitoring included COD removal efficiency, methane production, and periodic determination of the specific methanogenic activity in the presence of acetate, propionate, butyrate, ethanol and H2/CO2. Quantitative image analysis allowed for information to be obtained on granular fragmentation/erosion and filaments release. During the first operational period, biogas production was mainly due to the hydrogenotrophic activity. However, after 40 days, the SAA steadily increased achieving a maximum value of 183713mL CH4@STP/gVSS.d. The onset of SAA recovery, granules breakdown and filaments release to the bulk occurred simultaneously. Further increase in SAA was accompanied by granular growth. In the last 25 days of operation, the size distribution was stable with more than 80% of projected area of aggregates corresponding to granules larger than 1mm (equivalent diameter). Confocal images from FISH hybridized sections of the granules showed that after SAA recovery, the granules developed an organized structure where an acidogenic/acetogenic external layer was apparent. Granular fragmentation and increase of filaments in the bulk, simultaneously with the increase in the acetoclastic activity are described for the first time and might represent a structural response of granular sludge to promote the optimal substrate uptake at minimal diffusion limitations.Fundação para a Ciência e a Tecnologia (FCT) - Grants SFRH/BD/3187/2000, SFRH/BD/13317/2003, Project POCTI/BIO/37934/2001 Grants SFRH/BD/3187/2000, SFRH/BD/13317/200

Monitoring granular stability in an industrial UASB reactor treating brewery wastewater

An industrial scale UASB reactor treating a brewery wastewater was monitored during several months, in order to assess the usefulness of a morphological parameter relating the filaments length to the projected area of aggregates, LfA, to anticipate washout problems. It was observed that a selective washout of filamentous bacterial forms could explain the decrease of the acetoclastic and hydrogenophilic activity observed in the trial period. This selective washout was predicted by a peak in LfA that occurred just before a steady increase in the effluent VSS values.Grant SFRH/BD/3187/2000.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI/BD/20325/99

Image analysis as a tool to recognize anaerobic granulation time

Image analysis techniques were used to monitor the development of anaerobic granules into an EGSB reactor fed with a synthetic substrate based on glucose (30-40% COD) and volatile fatty acids (70-60% COD) during 400 days. Two types of objects were recognized and quantified: aggregates and free filaments. The aggregates were classified into five ranges based on the equivalent diameter: smaller than 11.3 mm (residual), between 11.3 and 35.7 mm, between 35.7 and 112.8 mm, between 112.8 and 356.8 mm and larger than 356.8 mm. For each size range the following parameters were measured: projected area, number of aggregates, width, length, perimeter, and convex envelop. For filaments, the measurements were: number of free filaments and total free filament length. Other parameters were calculated based upon this data: average equivalent diameter, percentage in number of aggregates, percentage in total area, fractal dimensions and a shape factor (P2A), based on the perimeter and on the projected area. Besides, a new parameter was defined in terms of the ratio of filament length to total floc area (LfA). It showed to be very sensitive to the changes in the morphology of the developing granules, and, based upon our granule definition, it was used to determine the granulation time (θg) in our system (120 days). The P_2A factor allowed the quantification of aggregates roughness and was much more sensitive than fractal dimensions. The study was complemented with measurements of specific methanogenic activity (SMA) of the granules in presence of acetate, propionate, butyrate, ethanol and a gas mixture of hydrogen/carbon dioxide.Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI/BD/20325/99 , SFRH/BD/3187/2000

Efeito da diluição na caracterização da biomassa de sistemas de tratamentos de efluentes por análise de imagem

O estudo da biomassa de sistemas de tratamento de efluentes por análise de imagem permite avaliar o seu estado de agregação assim como acompanhar as modificações por ela sofrida quando submetida a choques orgânicos ou hidráulicos. Certos processos de tratamento trabalham com uma forte concentração em biomassa, o que obriga a uma diluição prévia. O presente estudo avalia o efeito desta diluição sobre a caracterização da biomassa aeróbia e anaeróbia (agregada sob a forma de flocos ou grânulos ou isolada). Os resultados obtidos indicam que este efeito varia segundo a origem e tipo da biomassa e o tipo de água utilizada na diluição. Foi observado que a diluição provoca inicialmente um aumento do tamanho dos flocos para em seguida provocar a sua ruptura. Devido a fortes variações sofridas sobretudo em relação à quantificação das bactérias filamentosas, recomenda-se a opção por uma diluição e sua manutenção até o fim do experimento.The study by image analysis of biomass in wastewater treatment systems allow us to evaluate its aggregation state and to relate its changes to changes in the process operation conditions. In some processes, a high biomass concentration is used and dilution is necessary for visualisation by optical microscopy. The effect of the dilution factor on the measured characteristics of aerobic and anaerobic bacteria (aggregated as flocs or granules, or isolated) has been investigated. The results indicate that the effect depends on the origin and type of biomass and on the kind of water used for dilution. Dilution induces initially an increase of the floc size. The flocs are disrupted when the dilution factor is high. Due to the observed variations on the quantification of filamentous bacteria, it is recommended to select initially an appropriate dilution factor and to maintain it for the whole experiment.Brasil. Ministério da Ciência e Tecnologia. Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Fundação para a Ciência e a Tecnologia (FCT).Embaixada de França em Portugal.Ministério da Ciência e Tecnologia. Instituto de Cooperação Científica e Tecnológica Internacional (ICCTI)

Quantitative image analysis for the characterization of microbial aggregates in biological wastewater treatment : a review

Quantitative image analysis techniques have gained an undeniable role in several fields of research during the last decade. In the field of biological wastewater treatment (WWT) processes, several computer applications have been developed for monitoring microbial entities, either as individual cells or in different types of aggregates. New descriptors have been defined that are more reliable, objective, and useful than the subjective and time-consuming parameters classically used to monitor biological WWT processes. Examples of this application include the objective prediction of filamentous bulking, known to be one of the most problematic phenomena occurring in activated sludge technology. It also demonstrated its usefulness in classifying protozoa and metazoa populations. In high-rate anaerobic processes, based on granular sludge, aggregation times and fragmentation phenomena could be detected during critical events, e.g., toxic and organic overloads. Currently, the major efforts and needs are in the development of quantitative image analysis techniques focusing on its application coupled with stained samples, either by classical or fluorescent-based techniques. The use of quantitative morphological parameters in process control and online applications is also being investigated. This work reviews the major advances of quantitative image analysis applied to biological WWT processes.The authors acknowledge the financial support to the project PTDC/EBB-EBI/103147/2008 and the grant SFRH/BPD/48962/2008 provided by Fundacao para a Ciencia e Tecnologia (Portugal)

Quantitative image analysis as a diagnostic tool for identifying structural changes of anaerobic granular sludge

Tese de Doutoramento em Engenharia Química e BiológicaIn this thesis quantitative image analysis techniques were applied to monitor structural changes of anaerobic sludge under different operating conditions. In a first experiment that lasted 177 days, the phenomenon of aggregation of dispersed sludge was quantitatively monitored through the recognition and quantification of aggregates and free filaments. Free filaments were defined as filaments completely buoyant in the medium possessing both extremities free and those with only one free extremity being the other attached to an aggregate. A parameter defined as the ratio between the total free filament length and the total aggregate area (LfA) proved to be sensitive to structural changes in the aggregation status of the anaerobic sludge. LfA allowed the recognition of the aggregation time and revealed to be a good indicator of granular sludge fragmentation and erosion which usually leads to washout in high rate reactors. In a second operation period, image analysis was applied to monitor morphological changes in the previously formed granules, induced by increases in the upflow velocity and in the organic loading rate. In a further work, the initial stages of granules formation in EGSB reactors was assessed, with glucose and also gelatine as primary substrates. Granule development was monitored by image analysis techniques and FISH combined with confocal laser scanning microscopy. Strength, density and settling velocity were the bulk properties analyzed. Supported by the results, it is proposed that instead of nucleating and growing as a dense conglomerate, anaerobic granules initially grow in a sparse conglomerate to a large size, and then increase in density to maturity. In a later work, four EGSB reactors were operated at different influent nitrate concentrations (0, 20, 60 and 100 mgN-NO3-/L). After about 20 days of operation, the granular sludge was characterized morphologically by quantitative image analysis. Also specific methanogenic activities and settling velocities were measured. LfA parameter, was sensitive to quantify the different surface morphology, induced by nitrate in the anaerobic granular sludge. In a final experiment and in order to study the recovery of the SAA in granular sludge, an EGSB reactor was operated with ethanol was fed as the sole carbon source, during a trial period of 106 days. Quantitative image analysis allowed for information to be obtained on granular fragmentation/erosion and filaments release. Granular fragmentation and increase of filaments in the bulk simultaneous with the increase in the acetoclastic activity are described for the first time and might represent a structural response of granular sludge to promote the optimal substrate uptake at minimal diffusion limitations.Nesta tese são aplicadas técnicas de análise quantitativa de imagem para monitorizar alterações estruturais de biomassa anaeróbia em diferentes condições operatórias. Numa primeira experiência de 177 dias monitorizou-se o fenómeno de agregação de biomassa anaeróbia dispersa, através do reconhecimento e quantificação de agregados e filamentos livres. Filamentos livres foram definidos como filamentos que estão completamente livres no meio e que portanto possuem as duas extremidades livres, e também os filamentos que têm apenas uma extremidade livre e a outra ligada a um agregado. Definiu-se uma parâmetro que representa a razão entre o comprimento total de filamentos livres e a área total dos agregados (LfA). LfA provou ser sensível a alterações do estado de agregação da biomassa, e permitiu identificar o tempo de granulação. Mostrou ainda ser um bom indicador de fenómenos de erosão e fragmentação granulares que estão normalmente associados ao “washout” em reactores de alta carga. Numa segunda experiência, as técnicas de análise de imagem descritas foram aplicadas na monitorização dos grânulos previamente formados e nas alterações estruturais induzidas por variações na velocidade superficial e na carga orgânica aplicada. Numa outra experiência foram monitorados os estágios iniciais da formação de grânulos, usando glucose e gelatina como substratos. O desenvolvimento dos grânulos foi monitorizado por técnicas de análise de imagem e por técnicas de hibridização “in-situ” de fluorescência, combinadas com microscopia confocal laser. Foram medidas características físicas dos grânulos tais como resistência mecânica, densidade e velocidade de sedimentação. Com base nos resultados obtidos é proposto um novo modelo de granulação, segundo o qual, em vez de haver uma nucleação inicial e o crescimento de um agregado denso, se formam inicialmente agregados pouco densos que crescem até um tamanho razoável e que então se tornam mais densos, numa fase de maturação. Numa outra experiência foi utilizada a técnica de análise de imagem para avaliar as alterações morfológicas induzidas pela presença de nitrato em quatro reactores EGSB, alimentados com 0, 20, 60 e 100 mgN-NO3-/L, respectivamente. Após 20 dias de operação a biomassa granular foi caracterizada em termos de actividade metanogénica, morfologia e velocidade d sedimentação. O parâmetro LfA foi sensível às alterações morfológicas dos agregados microbianos, induzidas pelo nitrato. Numa experiência final foi estudada a recuperação da actividade metanogénica acetoclastica em biomassa anaeróbia granular durante 106 dias, num reactor EGSB alimentado com etanol. Através da análise quantitativa de imagem foi possível quantificar fenómenos de fragmentação erosão e libertação de filamentos durante o processo e relacioná-los com o aumento de actividade metanogénica acetoclastica observada durante o período experimental. Sugeriu-se que as alterações estruturais dos grânulos observadas, nomeadamente a libertação de filamentos e a fragmentação poderão ser um mecanismo de resposta dos agregados no sentido de promover a máxima assimilação de substrato minimizando problemas de limitações difusionais.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/3187/2000.GRICES/Danish Rector's Conference