Respirometric techniques applied to aerobic microbial processes

Tese de doutoramento em Chemical and Biological Engineeringerobic microbial processes are extremely important in environmental biotechnology, namely in biological wastewater treatment where activated sludge process represents nowadays the most widespread technology for wastewater purification. The availability of reliable, cheap and versatile real-time monitoring tools providing information on the biological activity is of crucial importance for monitoring and control of bioprocesses, avoiding possible operational troubles through early detection of problems. This thesis aimed to develop and apply respirometric techniques for the estimation of kinetic and stoichiometric parameters in different systems with increasing complexity. The first process – nitrification, was chosen for its simplicity. In this process, substrate consumption is related only with growth and energy, no storage phenomena occur. The ASM1 model was used to fit the respirometric data, and an in situ pulse respirometric method was validated for the determination of kinetic and stoichiometric parameters of the system at steady state: the maximum exogenous oxygen uptake rate was determined respirometrically [61.15 (4.09) mg O2 L-1 h-1] through the injection of increasing substrate concentration pulses; the biomass growth yield was estimated from respirometric data and by the traditional COD (Chemical Oxygen Demand) mass balance method, and both methods gave similar values [0.10 (0.07) and 0.09 (0.04) g X-COD g NOD-1, respectively]; the affinity constant was indirectly estimated after fitting the ascending part of the respirogram to a theoretical model, and an average value of 0.48 (0.08) mg NH4-N L-1 was obtained. Model adjustment was successfully applied to a portion of the respirogram, but not to the complete respirogram. It was concluded that a more complex model, taking into account biological and electrode response time, should give better correlation. Additionally, two methods for the determination of the oxygen volumetric mass transfer coefficient were tested and it was concluded that the dynamic method was the most adequate. The results obtained allowed to establish the basic pulse respirometric methodology, and also the best method for estimating the oxygen mass transfer coefficient. Then a Pseudomonas putida pure culture system was chosen to test the applicability of pulse respirometry on biomass with storage ability ASM3 was used for fitting the obtained data. The model included terms which took into account the biological and electrode response time, thus the entire respirogram could be successfully used for model fitting. The pulse respirometric method was validated for estimation of kinetic and stoichiometric parameters in pure cultures by comparison with the traditional chemostat method. No significance difference was observed between parameters estimated by chemostat and respirometric methods: biomass growth yield was 0.41(0.05) and 0.51 (0.04) g COD-X g COD-S-1; affinity constant was 4.86 (0.70) and 5.13 (1.99) mg COD-S L-1; maximum specific growth rate was 0.20 (0.05) and 0.16 (0.01) h-1, with chemostat and respirometry respectively. Pulse respirometry was then applied to a continuous suspended activated sludge process, fed with a complex synthetic medium. In this stage, a multiple pulses respirometric method was tested and validated by comparison with chemostat method and with ASM1 model fitting: biomass growth yield was in the range of 0.37 – 0.76, and 0.37 – 0.65 g COD-X g COD-S-1; maintenance coefficient was 0.012 (0.012) and 0.010 (0.006) h-1, by COD mass balance and by respirometry, respectively; affinity constant and maximum specific oxygen consumption rate were estimated with the multiple concentration pulses respirometric method to be 15.5 (2.4) mg COD-S L-1 and 0.12 (0.01) h-1, respectively. Considering the assessment and comparison of the experimental and calculation methods, it was concluded that the estimation of kinetic and stoichiometric parameters in mixed aerobic cultures should preferentially be performed by using respirometric techniques, being the most adequate method the multiple concentration pulses injection method, with several advantages such as a simpler experimental data interpretation, and results with better confidence. The developed multiple pulses respirometric method was finally applied to an aerobic granular system. The method proved to be adequate for parameter estimation on this system, and allowed the successful monitoring of aerobic granulation. In a short time and using low cost equipment, the method allowed an exhaustive characterisation of the process in real-time through the determination of six central parameters: (i) biomass growth yield, (ii) specific endogenous respiration rate, (iii) substrate affinity constant, (iv) maximum specific oxygen consumption rate, (v) maximum specific substrate consumption rate, and (vi) maximum specific growth rate. The pulse respirometric method presented the advantage of the determined parameters being those actually prevailing in the system under actual operating conditions, i.e. apparent parameters, which is of major interest for control and process operation. At steady state the biomass growth yield was estimated to be 0.6 g COD-X g COD-S-1, the specific endogenous respiration rate was 0.1 g O2 g COD-X-1 d-1, the affinity constant was approximately 20 mg COD-S L-1, maximum specific oxygen consumption rate and maximum specific substrate consumption rate were 0.06 g O2 g COD-X-1 h-1 and 0.17 g COD-S g CODX- 1 h-1, respectively, and the maximum specific growth rate was roughly 2.5 d-1. The potential of the proposed multiple concentration pulses respirometric method was investigated for monitoring aerobic granular sludge systems, and controlling aeration in an efficient mode. Additionally the multiple concentration pulses respirometric method was applied in two aerobic granular sludge systems operated under different aeration rates (5.0 and 6.6 L min-1), and allowed to assess the influence of shear stress on the biomass kinetic and stoichiometric parameters: the biomass growth yield was higher for the sludge cultivated under higher shear stress [0.6 (0.02) versus 0.5 (0.02) g COD-X g COD-S-1 at lower shear stress]; biomass subjected to the higher shear stress presented a higher substrate affinity constant [16.4 (2.6) and 9.1 (0.2) mg COD-S L-1, at higher and lower shear stress, respectively]; and also a higher maximum specific substrate consumption rate [5.4 (0.9) and 2.5 (0.5) g COD-S g COD-X d-1, at higher and lower shear stress, respectively] at the end of the aerobic granulation process. The global conclusion withdrawn from this thesis is that respirometry, especially pulse respirometry, is a valid and promising technique for kinetic and stoichiometric characterisation of aerobic microbial processes, whether these are pure or mixed cultures, and suspended or aggregated cultures. A respirometric method was developed, the multiple concentration pulses method, which allows an exhaustive characterisation of aerobic microbial processes in a short time period, using low cost material and requiring low computational power.Os processos microbianos aeróbios são extremamente importantes em biotecnologia ambiental, nomeadamente nos sistemas biológicos de tratamento de águas residuais onde o processo de lamas activadas representa actualmente a tecnologia mais generalizada de purificação de água. A disponibilidade de ferramentas de monitorização em tempo real confiáveis, baratas e versáteis, que forneçam informações sobre a actividade biológica, é de importância crucial para a monitorização e controlo de processos biológicos, evitando problemas operacionais através da detecção precoce de problemas. Esta tese teve como objectivo desenvolver e aplicar técnicas respirométricas para a determinação de parâmetros cinéticos e estequiométricos em diferentes sistemas com complexidade crescente. O primeiro processo – nitrificação, foi escolhido pela sua simplicidade. Neste processo, o consumo de substrato está relacionado apenas com o crescimento e produção de energia, nenhum fenómeno de armazenamento de substrato ocorre e um método respirométrico in situ foi validado para a determinação de parâmetros cinéticos e estequiométricos em sistemas em estado estacionário: a taxa máxima específica de consumo de oxigénio foi determinada respirometricamente [61.15 (4.09) mg O2 L-1 h-1] através da injecção de pulsos de concentração de substrato crescente; o rendimento de crescimento da biomassa foi estimado a partir de dados respirométricos e a partir do método tradicional de balanço de massa à CQO (Carência Química de Oxigénio) e ambos os métodos deram resultados similares [0.10 (0.07) e 0.09 (0.04) g X-COD g NOD-1, respectivamente]; a constante de afinidade do substrato foi determinada indirectamente após ajuste de um modelo teórico à porção ascendente do respirograma e um valor médio de 0.48 (0.08) mg NH4-N L-1 foi determinado. O ajuste de um modelo foi bem sucedido com uma porção do respirograma, mas não com o respirograma inteiro. Foi concluído que um modelo mais complexo, que tome em consideração os tempos de resposta do eléctrodo e da biomassa, deverá ajustar melhor ao respirograma. Adicionalmente, dois métodos para a determinação do coeficiente volumétrico de transferência de massa do oxigénio foram testados e concluiu-se que o método mais adequado é o método dinâmico. Os resultados obtidos permitiram estabelecer os princípios básicos da metodologia da respirometria de pulsos e o melhor método para determinação do coeficiente volumétrico de transferência de massa do oxigénio. Seguidamente um sistema de cultura pura de Pseudomonas putida foi seleccionado para testar a aplicabilidade da respirometria de pulso numa biomassa com capacidade de armazenar substrato. O modelo ASM3 foi usado para ajuste aos dados experimentais. O modelo incluiu termos que consideravam os tempos de resposta do eléctrodo e da biomassa, assim o respirograma inteiro pôde ser usado para ajuste do modelo. O método respirométrico de pulsos foi validado para a determinação de parâmetros cinéticos e estequiométricos em culturas puras por comparação com o método tradicional de quimiostato, visto que as diferenças dos parâmetros determinados pelos dois métodos não foram significativas: o rendimento em biomass 0.41 (0.05) e 0.51 (0.04) g COD-X g COD-S-1; a constante de afinidade do substrato 4.86 (0.70) e 5.13 (1.99) mg COD-S L-1; a taxa máxima específica de crescimento 0.20 (0.05) e 0.16 (0.01) h-1, com o método do quimiostato e respirométrico, respectivamente. A respirometria de pulso foi depois aplicada a um processo de lamas activadas suspensas alimentado com meio sintético complexo. Nesta etapa, um método respirométrico de pulsos múltiplos foi testado e validado por comparação com o método do quimiostato e com ajuste do modelo ASM1: o rendimento em biomassa estava na gama 0.37 – 0.76 e 0.37 – 0.65 g COD-X g COD-S-1; o coeficiente de manutenção 0.012 (0.012) e 0.010 (0.006) h-1, por balanço mássico de CQO e por respirometria, respectivamente; a constante de afinidade do substrato e a taxa máxima específica de consumo de oxigénio foram determinadas com o método respirométrico de pulsos múltiplos obtendo-se os valores 15.5 (2.4) mg COD-S L-1 e 0.12 (0.01) h-1, respectivamente. Considerando a acessibilidade e comparação dos métodos experimentais e de cálculo, foi concluído que a determinação de parâmetros cinéticos e estequiométricos em culturas mistas aeróbias deve ser preferencialmente feita usando técnicas respirométricas, sendo o método respirométrico de pulsos múltiplos o mais adequado com várias vantagens tais como uma mais simples interpretação dos dados experimentais e obtenção de resultados mais exactos. O método respirométrico de pulsos múltiplos desenvolvido foi finalmente aplicado a um sistema de grânulos aeróbios. O método mostrou ser adequado para determinação de parâmetros neste tipo de sistema e permitiu a monitorização do processo de granulação aeróbia. Num curto tempo e usando equipamento de baixo custo o método permitiu a caracterização exaustiva do processo em tempo real através da determinação de seis parâmetros centrais: (i) rendimento em biomassa; (ii) taxa específica de respiração endógena; (iii) constante de afinidade do substrato; (iv) taxa máxima específica de consumo de oxigénio; (v) taxa máxima específica de consumo de substrato e (vi) taxa máxima específica de crescimento. O método respirométrico de pulsos múltiplos apresentou ainda a vantagem de fornecer parâmetros relacionados com os que realmente estão a ocorrer no sistema sobre as reais condições operacionais, i.e. os parâmetros aparentes, o que é de maior interesse para controlo e operação. Em estado estacionário o rendimento em biomassa foi estimado como sendo de 0.6 g COD-X g COD-S-1, a taxa específica de respiração endógena 0.1 g O2 g COD-X-1 d-1, a constante de afinidade do substrato aproximadamente 20 mg COD-S L-1, a taxa máxima específica de consumo de oxigénio e a taxa máxima específica de consume de substrato 0.06 g O2 g COD-X-1 h-1 e 0.17 g COD-S g COD-X-1 h-1, respectivamente, e a taxa máxima específica de crescimento 2.5 d-1. O potencial do método respirométrico de pulsos múltiplos proposto foi investigado para monitorização de sistemas de grânulos aeróbios e para o controlo eficiente da taxa de arejamento. Adicionalmente, o método respirométrico de pulsos múltiplos foi aplicado a dois sistemas de grânulos aeróbios operados a diferentes taxas de arejamento (5.0 e 6.6 L min-1), permitindo verificar a influência da tensão de corte nos parâmetros cinéticos e estequiométricos: o rendimento em biomassa foi maior na biomassa cultivada na tensão de corte mais alta [0.6 (0.02) versus 0.5 (0.02) g COD-X g COD-S-1 a tensão de corte mais baixa]; a biomassa sujeita a uma tensão de corte mais elevada apresentou uma constante de afinidade do substrato mais alta [16.4 (2.6) e 9.1 (0.2) mg COD-S L-1, a tensão de corte mais alta e mais baixa, respectivamente] e também uma taxa máxima específica de consumo de substrato mais alta [5.4 (0.9) e 2.5 (0.5) g COD-S g COD-X d-1, a tensão de corte mais alta e mais baixa, respectivamente] no fim do processo de granulação aeróbia. A conclusão global retirada desta tese é que a respirometria, especialmente a respirometria de pulsos, é uma técnica válida e promissora para a caracterização cinética e estequiométrica de processos aeróbios, quer sejam culturas puras ou mistas, culturas suspensas ou agregadas. Um método respirométrico foi desenvolvido, o método respirométrico de pulsos múltiplos, que permite a caracterização exaustiva de processos microbianos aeróbios num curto tempo, usando equipamento de baixo custo e requerendo baixo poder computacional

Aerobic granulation kinetics, stoiquiometrics andmorphological assessment

In the present work, aerobic granulation of activated sludge was achieved in a sequencing batch airlift reactor (SBAR) fed with acetate as sole carbon source. Aerobic granulation process was followed by the assessment of the biomass kinetic and stoichiometric parameters through respirometric pulse experiments, and by the assessment of aggregates morphology through quantitative image analysis. This study contributes for a better understanding of the aerobic granulation process, introducing tools for aerobic granular biomass characterisation

Kinetic, stoichiometric, and morphological assessment of aerobic granulation in a sequencing batch airlift reactor

Aerobic granular sludge has recently become a promising environmental biotechnology process for wastewater treatment. Understanding the aerobic granulation process in all its facets is, therefore, of major importance. In the present work, aerobic granulation of activated sludge was achieved in a sequencing batch airlift reactor (SBAR) fed with acetate as sole carbon source. Aerobic granulation process was followed by the assessment of the biomass kinetics and stoichiometrics through respirometric pulse experiments, and by the assessment of aggregates morphology through image analysis. The SBAR was operated in 4 hour cycles, with 2 minutes settling time that promoted the selection of biomass with a minimum settling velocity of 11 m/h. The average COD removal efficiency was always above 90 %. Biomass concentration increased from 2.4 ± 1.3 g MLVSS/L to 10.4 ± 3.5 g MLVSS/L. Biomass density increased from 3.4 ± 0.3 g/L to a maximum of 15.8 ± 1.4 g/L. Compact aggregates with granular characteristics were identified after 4 days of operation. Average diameter increased from 0.47 ± 0.01 mm to 1.3 ± 0.05 mm (Figure 1). Respirometric experiments were performed to total biomass, and, separately, to aggregates with diameter bigger than 0.25 mm, designated as granular biomass. Stoichiometric results showed a significant difference between the apparent growth yields and the true growth yields of the granular sludge, indicating that granular biomass was storing substrate at a significant extent likely as a response to the alternate feast-famine periods typical in SBR reactors. Substrate affinity constant (KS) of the granules were higher than that of the total biomass, 43.4 and 31.3 h-1 respectively, this was attributed to the substrate diffusion through the granules. The predominance of granular biomass in the reactor was shown respirometrically by the significant contribution of the granular sludge to the total oxygen uptake rate (OUR). This study contributes for a better understanding of the aerobic granulation process, focussing on the kinetic, stoichiometric, and morphological characteristics

Assessment of the aerobic granulation process through quantitative image analysis

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/1096

Cognitive appraisal in fish: stressor predictability modulates the physiological and neurobehavioural stress response in sea bass

The role of cognitive factors in triggering the stress response is well established in humans and mammals (aka cognitive appraisal theory) but very seldom studied in other vertebrate taxa. Predictability is a key factor of the cognitive evaluation of stimuli. In this study, we tested the effects of stressor predictability on behavioral, physiological and neuromolecular responses in the European sea bass (Dicentrarchus labrax). Groups of four fish were exposed to a predictable (signalled) or unpredictable (unsignalled) stressor. Stressor predictability elicited a lower behavioural response and reduced cortisol levels. Using the expression of immediate early genes (c-fos, egr-1, bdnf and npas4) as markers of neuronal activity, we monitored the activity of three sea bass brain regions known to be implicated in stressor appraisal: the dorsomedian telencephalon, Dm (putative homologue of the pallial amygdala); and the dorsal (Dld) and ventral (Dlv) subareas of the dorsolateral telencephalon (putative homologue of the hippocampus). The activity of both the Dm and Dlv significantly responded to stressor predictability, suggesting an evolutionarily conserved role of these two brain regions in information processing related to stressor appraisal. These results indicate that stressor predictability plays a key role in the activation of the stress response in a teleost fish, hence highlighting the role of cognitive processes in fish stress.info:eu-repo/semantics/publishedVersio

20 years of Portuguese drug policy - developments, challenges and the quest for human rights

Portugal decriminalized the public and private use, acquisition, and possession of all drugs in 2000; adopting an approach focused on public health rather than public-order priorities. Arguing that the Portuguese Drug Policy Model has not proven influential enough to emancipate drug use from the stigma that associates it either with crime or pathology, this article critically discusses the developments and current challenges the Portuguese drug policy confronts, namely the growing diversity of drug use patterns observed in Portugal as well as in Europe. To this end, international and national legal instruments concerning drugs and official local data were analysed. Despite encouraging results, conclusions indicate that these policies are marked by contradictions and ambiguities that have permeated its history since the very beginning, and modest ambitions, particularly regarding the implementation of harm reduction measures. Moreover, the polemical Supreme Court judgment that reestablished, in 2008, drug use as a crime when the quantities at play exceeded those required for an average individual's use for 10 days, might have impacted the landscape of drug use penalization. The last decade saw an increase of punitiveness targeted at drug users, including criminal sentences of jail terms. We finish with some suggestions that could be employed in the practical application of drug policy

Microalgae-bacterial granular sludge systems - on the road for more sustainable processes in the aquaculture sector

With population growth and stagnation of capture fisheries, the aquaculture sector has been challenged to achieve remarkable production targets to meet the ever-increasing fish demand. However, land-based aquaculture industries need to capture high water volumes from nearby water bodies to ensure an adequate production and, consequently, high wastewater volumes, containing organic carbon, nutrients, and often contaminants of emerging concern, are produced. If not properly handled, aquaculture effluents pose a threat to receiving aquatic ecosystems. The rapid expansion of these industries, facing the increased demand for food worldwide, is only possible if more sustainable practices are adopted. To face the current water shortages and protect water resources, the development of environmentally friendly treatment systems that allow water recirculation is of utmost importance. This work aimed to develop a microalgae-bacteria granular sludge system able to efficiently treat marine aquaculture effluents so they can meet the requirements for recirculation. A photo-sequencing batch reactor was inoculated with a phototrophic microbial consortium obtained from water streams in a marine aquaculture facility and was fed with wastewater mimicking marine aquaculture streams. The aggregation of the microbial biomass occurred rapidly and, on day-21, ca. 49% of the total reactor biomass was in the form of granules. The system exhibited high and stable organic carbon removal (>80%), even when florfenicol, an antibiotic widely used in aquaculture, was present in the wastewater. Concerning the nitrogen content, a high-chemical quality effluent was obtained, complying with ammonium, nitrite, and nitrate concentrations for water recirculation within a marine aquaculture farm, even in periods where florfenicol was present in the wastewater. In addition, the dissolved oxygen levels in the treated effluents where within the ideal range for fish growth thus reducing the need for oxygenation and, consequently, the farms operational costs. Additionally, the coexistence of microalgae and bacteria within the granules allowed to treat wastewater at low air flow rates potentially reducing the energy needed for system’s aeration. Microalgae-bacterial granular sludge systems can contribute for the aquaculture sector sustainability as they enable to reduce energy and water usage whilst ensuring environmental protection.info:eu-repo/semantics/publishedVersio

Tamoxifen and estradiol interact with the flavin mononucleotide site of complex I leading to mitochondrial failure

This study evaluated the action of tamoxifen and estradiol on the function of isolated liver mitochondria. We observed that although tamoxifen and estradiol per se did not affect mitochondrial complexes II, III, or IV, complex I is affected, this effect being more drastic (except for state 4 of respiration) when mitochondria were coincubated with both drugs. Furthermore, using two respiratory chain inhibitors, rotenone and diphenyliodonium chloride, we identified the flavin mononucleotide site of complex I as the target of tamoxifen and/or estradiol action(s). Tamoxifen (25 microm) per se induced a significant increase in hydrogen peroxide production and state 4 of respiration. Additionally, a significant decrease in respiratory control ratio, transmembrane, and depolarization potentials were observed. Estradiol per se decreased carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)-stimulated respiration, state 3 of respiration, and respiratory control ratio and increased lag phase of repolarization. With the exception of state 4 of respiration whose increase induced by tamoxifen was reversed by the presence of estradiol, the effects of tamoxifen were highly exacerbated when estradiol was present. We observed that 10 microm tamoxifen in the presence of estradiol affected mitochondria significantly by decreasing FCCP-stimulated respiration, state 3 of respiration, respiratory control ratio, and ADP depolarization and increasing the lag phase of repolarization. All of the deleterious effects induced by 25 microm tamoxifen were highly exacerbated in the presence of estradiol. Furthermore, we observed that the effects of both compounds were independent of estrogen receptors because the pure estrogen antagonist ICI 182,780 did not interfere with tamoxifen and/or estradiol detrimental effects. Altogether, our data provide a mechanistic explanation for the multiple cytotoxic effects of tamoxifen including its capacity to destroy tamoxifen-resistant breast cancer cells in the presence of estradiol. This new piece of information provides a basis for the development of new and promising anticancer therapeutic strategie