Investigation of polyhydroxyalkanoate production by an activated sludge microbial consortium treating artificial dairy wastewater

Petrochemical plastics/polymers are a common feature of day to day living as they occur in packaging, furniture, mobile phones, computers, construction equipment etc. However, these materials are produced from non-renewable materials and are resistant to microbial degradation in the environment. Considerable research has therefore been carried out into the production of sustainable, biodegradable polymers, amenable to microbial catabolism to CO2 and H2O. A key group of microbial polyesters, widely considered as optimal replacement polymers, are the Polyhydroxyalkaonates (PHAs). Primary research in this area has focused on using recombinant pure cultures to optimise PHA yields, however, despite considerable success, the high costs of pure culture fermentation have thus far hindered the commercial viability of PHAs thus produced. In more recent years work has begun to focus on mixed cultures for the optimisation of PHA production, with waste incorporations offering optimal production cost reductions. The scale of dairy processing in Ireland, and the high organic load wastewaters generated, represent an excellent potential substrate for bioconversion to PHAs in a mixed culture system. The current study sought to investigate the potential for such bioconversion in a laboratory scale biological system and to establish key operational and microbial characteristics of same. Two sequencing batch reactors were set up and operated along the lines of an enhanced biological phosphate removal (EBPR) system, which has PHA accumulation as a key step within repeated rounds of anaerobic/aerobic cycling. Influents to the reactors varied only in the carbon sources provided. Reactor 1 received artificial wastewater with acetate alone, which is known to be readily converted to PHA in the anaerobic step of EBPR. Reactor 2 wastewater influent contained acetate and skim milk to imitate a dairy processing effluent. Chemical monitoring of nutrient remediation within the reactors as continuously applied and EBPR consistent performances observed. Qualitative analysis of the sludge was carried out using fluorescence microscopy with Nile Blue A lipophillic stain and PHA production was confirmed in both reactors. Quantitative analysis via HPLC detection of crotonic acid derivatives revealed the fluorescence to be short chain length Polyhydroxybutyrate, with biomass dry weight accumulations of 11% and 13% being observed in reactors 1 and 2, respectively. Gas Chromatography-Mass Spectrometry for medium chain length methyl ester derivatives revealed the presence of hydroxyoctanoic, -decanoic and -dodecanoic acids in reactor 1. Similar analyses in reactor 2 revealed monomers of 3-hydroxydodecenoic and 3-hydroxytetradecanoic acids. Investigation of the microbial ecology of both reactors as conducted in an attempt to identify key species potentially contributing to reactor performance. Culture dependent investigations indicated that quite different communities were present in both reactors. Reactor 1 isolates demonstrated the following species distributions Pseudomonas (82%), Delftia acidovorans (3%), Acinetobacter sp. (5%) Aminobacter sp., (3%) Bacillus sp. (3%), Thauera sp., (3%) and Cytophaga sp. (3%). Relative species distributions among reactor 2 profiled isolates were more evenly distributed between Pseudoxanthomonas (32%), Thauera sp (24%), Acinetobacter (24%), Citrobacter sp (8%), Lactococcus lactis (5%), Lysinibacillus (5%) and Elizabethkingia (2%). In both reactors Gammaproteobacteria dominated the cultured isolates. Culture independent 16S rRNA gene analyses revealed differing profiles for both reactors. Reactor 1 clone distribution was as follows; Zooglea resiniphila (83%), Zooglea oryzae (2%), Pedobacter composti (5%), Neissericeae sp. (2%) Rhodobacter sp. (2%), Runella defluvii (3%) and Streptococcus sp. (3%). RFLP based species distribution among the reactor 2 clones was as follows; Runella defluvii (50%), Zoogloea oryzae (20%), Flavobacterium sp. (9%), Simplicispira sp. (6%), Uncultured Sphingobacteria sp. (6%), Arcicella (6%) and Leadbetterella bysophila (3%). Betaproteobacteria dominated the 16S rRNA gene clones identified in both reactors. FISH analysis with Nile Blue dual staining resolved these divergent findings, identifying the Betaproteobacteria as dominant PHA accumulators within the reactor sludges, although species/strain specific allocations could not be made. GC analysis of the sludge had indicated the presence of both medium chain length as well short chain length PHAs accumulating in both reactors. In addition the cultured isolates from the reactors had been identified previously as mcl and scl PHA producers, respectively. Characterisations of the PHA monomer profiles of the individual isolates were therefore performed to screen for potential novel scl-mcl PHAs. Nitrogen limitation driven PHA accumulation in E2 minimal media revealed a greater propensity among isoates for mcl-pHA production. HPLC analysis indicated that PHB production was not a major feature of the reactor isolates and this was supported by the low presence of scl phaC1 genes among PCR screened isolates. A high percentage distribution of phaC2 mcl-PHA synthase genes was recorded, with the majority sharing high percentage homology with class II synthases from Pseudomonas sp. The common presence of a phaC2 homologue was not reflected in the production of a common polymer. Considerable variation was noted in both the monomer composition and ratios following GC analysis. While co-polymer production could not be demonstrated, potentially novel synthase substrate specificities were noted which could be exploited further in the future.

Impact of carbon/nitrogen feeding strategy on polyhydroxyalkanoates production using mixed microbial cultures

Polyhydroxyalkanoates (PHA) production using mixed microbial cultures (MMC) requires a multi-stage process involving the microbial selection of PHA-storing microorganisms, typically operated in sequencing batch reactors (SBR), and an accumulation reactor. Since low-cost renewable feedstocks used as process feedstock are often nitrogen-deficient, nutrient supply in the selection stage is required to allow for microbial growth. In this context, the possibility to uncouple nitrogen supply from carbon feeding within the SBR cycle has been investigated in this study. Moreover, three different COD:N ratios (100:3.79, 100:3.03 and 100:2.43) were tested in three different runs which also allowed the study of COD:N ratio on the SBR performance. For each run, a synthetic mixture of acetic and propionic acids at an overall organic load rate of 8.5 gCOD L-1 d-1 was used as carbon feedstock, whereas ammonium sulfate was the nitrogen source in a lab-scale sequence batch reactor (SBR) with 1 L of working volume. Besides, a sludge retention time (SRT) of 1 d was used as well as a 6 h cycle length. The uncoupled feeding strategy significantly enhanced the selective pressure towards PHA-storing microorganisms, resulting in a two-fold increase in the PHA production (up to about 1.3 gCOD L-1). A high storage response was observed for the two runs with the COD:N ratios (gCOD:gN) of 100:3.79 and 100:3.03, whereas the lowest investigated nitrogen load resulted in very poor performance in terms of polymer production. In fact, strong nitrogen limitation caused fungi to grow and a very poor storage ability by microorganisms that thrived in those conditions. The COD:N ratio also affected the polymer composition, indeed the produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) showed a variable HV content (1-20 %, w/w) among the three runs, lessening as the COD:N increased. This clearly suggests the possibility to use the COD:N ratio as a tool for tuning polymer properties regardless the composition of the feedstock.

Influence of Major Operational Parameters on Aerobic Granulation for the Treatment of Wastewater

Effective solids-liquid separation is the basic concept of any wastewater treatment system. Biological treatment methods involve microorganisms for the treatment of wastewater. Conventional activated sludge process (ASP) poses the problem of poor settleability and hence require a large footprint. Biogranulation is an effective biotechnological process which can overcome the drawbacks of conventional ASP to a great extent. Aerobic granulation represents an innovative cell immobilization strategy in biological wastewater treatment. Aerobic granules are selfimmobilized microbial aggregates that are cultivated in sequencing batch reactors (SBRs). Aerobic granules have several advantages over conventional activated sludge flocs such as a dense and compact microbial structure, good settleability and high biomass retention. For cells in a culture to aggregate, a number of conditions have to be satisfied. Hence aerobic granulation is affected by many operating parameters. The organic loading rate (OLR) helps to enrich different bacterial species and to influence the size and settling ability of granules. Hence, OLR was argued as an influencing parameter by helping to enrich different bacterial species and to influence the size and settling ability of granules. Hydrodynamic shear force, caused by aeration and measured as superficial upflow air velocity (SUAV), has a strong influence and hence it is used to control the granulation process. Settling time (ST) and volume exchange ratio (VER) are also two key influencing factors, which can be considered as selection pressures responsible for aerobic granulation based on the concept of minimal settling velocity. Hence, these four parameters - OLR, SUAV, ST and VER- were selected as major influencing parametersfor the present study. Influence of these four parameters on aerobic granulation was investigated in this work

Control and optimization of an SBR for nitrogen removal: from model calibration to plant operation

En aquesta tesis s'ha desenvolupat un sistema de control capaç d'optimitzar el funcionament dels Reactors Discontinus Seqüencials dins el camp de l'eliminació de matèria orgànica i nitrogen de les aigües residuals. El sistema de control permet ajustar en línia la durada de les etapes de reacció a partir de mesures directes o indirectes de sondes. En una primera etapa de la tesis s'ha estudiat la calibració de models matemàtics que permeten realitzar fàcilment provatures de diferents estratègies de control. A partir de l'anàlisis de dades històriques s'han plantejat diferents opcions per controlar l'SBR i les més convenients s'han provat mitjançant simulació. Després d'assegurar l'èxit de l'estratègia de control mitjançant simulacions s'ha implementat en una planta semi-industrial. Finalment es planteja l'estructura d'uns sistema supervisor encarregat de controlar el funcionament de l'SBR no només a nivell de fases sinó també a nivell cicle.

Reatores anaeróbios operados em batelada sequencial, seguidos de lagoas de polimento, para o tratamento de águas residuárias de suinocultura. Parte I: produção de metano e remoção de DQO e de sólidos suspensos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reatores anaeróbios operados em batelada sequencial seguidos de lagoas de polimento para o tratamento de águas residuárias de suinocultura. Parte II: remoção de nutrientes e coliformes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tratamento de esgoto sanitário utilizando reatores anaeróbios operados em bateladas sequenciais (escala piloto)

The performances of two anaerobic sequencing batch reactors (1.2 m 3) containing biomass immobilized in inert support and as granular sludge in the treatment of domestic sewage from the Campus of São Carlos-University of São Paulo were evaluated. The experimental phase lasted seventy days. During this period, the reactors presented quite similar performances in respect to COD and total suspended solids removal, achieving average efficiencies of approximately 60% and 75%, respectively. The analysis using molecular biology techniques on biomass samples taken at 35 th and 70 th showed differences in the bacterial community in the reactors indicating that the type of biomass immobilization selected the populations differently. A higher similarity was found for the Archaea domain probably because these microorganisms utilize specific substrates formed at the end of the anaerobic process.

Comparação da eficiência de reatores aeróbios com paralisações escalonadas de aeração em diferentes concentrações de matéria orgânica no tratamento de águas residuárias do processamento de farinha de mandioca

Cassava wastewater, generated during cassava processing, is a highly polluting and toxic waste. This study aimed to assess the relationship of cassava treatment efficiency in two aerobic sequencing batch reactors, cylindrical plastics (31cm height x 9cm diameter), with 2.0 liters capacity and 1.5 liters of work volume, a ratio of diameter and height of the liquid 1:2.5, with a running time of 24 hours and stoppage aeration for 16 hours with average feed of 2,500 and 6,000 mg COD. L -1. COD, pH, SVI and F/M were analyzed. The results obtained in the two reactors showed an average reduction of 94.1% and 76.8% organic content, respectively; pH values showed a rising in the output pH values compared to entry ones; SVI values obtained: 54.1 mL/g, 99.3 mL/g, respectively in a F/M ratio values worked out of 0.4 d-1 and 1,1 d-1 , respectively. The results demonstrated that the process has generated considerable saving in energy consumption compared to traditional continuous systems, was efficient and did not affect the efficiency overly of the reactor.

Implementation of enhanced biological phosphorus removal (ebpr) wastewater treatment processes enriched with different microbial communities

The EBPR (Enhanced Biological Phosphorus Removal) is a type of secondary treatment in WWTPs (WasteWater Treatment Plants), quite largely used in full-scale plants worldwide. The phosphorus occurring in aquatic systems in high amounts can cause eutrophication and consequently the death of fauna and flora. A specific biomass is used in order to remove the phosphorus, the so-called PAOs (Polyphosphate Accumulating Organisms) that accumulate the phosphorus in form of polyphosphate in their cells. Some of these organisms, the so-called DPAO (Denitrifying Polyphosphate Accumulating Organisms) use as electron acceptor the nitrate or nitrite, contributing in this way also to the removal of these compounds from the wastewater, but there could be side reactions leading to the formation of nitrous oxides. The aim of this project was to simulate in laboratory scale a EBPR, acclimatizing and enriching the specialized biomass. Two bioreactors were operated as Sequencing Batch Reactors, one enriched in Accumulibacter, the other in Tetrasphaera (both PAOs): Tetrasphaera microorganisms are able to uptake aminoacids as carbon source, Accumulibacter uptake organic carbon (volatile fatty acids, VFA). In order to measure the removal of COD, phosphorus and nitrogen-derivate compounds, different analysis were performed: spectrophotometric measure of phosphorus, nitrate, nitrite and ammonia concentrations, TOC (Total Organic Carbon, measuring the carbon consumption), VFA via HPLC (High Performance Liquid Chromatography), total and volatile suspended solids following standard methods APHA, qualitative microorganism population via FISH (Fluorescence In Situ Hybridization). Batch test were also performed to monitor the NOx production. Both specialized populations accumulated as a result of SBR operations; however, Accumulibacter were found to uptake phosphates at higher extents. Both populations were able to remove efficiently nitrates and organic compounds occurring in the feeding. The experimental work was carried out at FCT of Universidade Nova de Lisboa (FCT-UNL) from February to July 2014.

Tratamento de água residuária parcialmente solúvel em reator anaeróbio em batelada seqüencial contendo biomassa imobilizada e agitação mecânica: análise da influência da intensidade de agitação e do tamanho de biopartícula

Neste trabalho de doutorado utilizou-se um reator anaeróbio em batelada seqüencial contendo biomassa imobilizada em matrizes cúbicas de espuma de poliuretano e agitação mecânica, com volume total de 5.5 L e volume útil de 4.5 L. A agitação do meio líquido foi realizada com quatro tipos de impelidores (turbina tipo hélice, lâminas planas, lâminas planas inclinadas e lâminas curvas, testados individualmente, sempre em número de 3), com 6 cm de diâmetro. A pesquisa foi realizada em quatro etapas experimentais. A etapa 1 objetivou determinar o tempo de mistura no reator para cada tipo de impelidor, ou seja, o tempo necessário para que o meio líquido ficasse totalmente homogêneo. A etapa 2 objetivou selecionar o tipo de impelidor e a respectiva intensidade de agitação que garantisse a minimização da resistência à transferência de massa externa no sistema. As intensidades de agitação testadas variaram de 200 a 1100 rpm, dependendo do tipo de impelidor. A etapa 3 foi realizada com tipo de impelidor e intensidade de agitação definidos na etapa 2, mas variando-se o tamanho da biopartícula (0,5, 1,0, 2,0 e 3,0 cm de aresta). O objetivo desta etapa foi selecionar o tamanho de biopartícula que minimizasse a resistência à transferência de massa interna. De posse das condições operacionais otimizadas (tipo de impelidor, intensidade de agitação e tamanho de partícula de suporte), a etapa 4 constituiu na aplicação das mesmas para o tratamento de um resíduo real, sendo escolhida água residuária de suinocultura. Na etapa 1, os resultados mostraram que os tempos de mistura para todos os tipos de impelidores foram desprezíveis em relação ao tempo total de ciclo. A etapa 2 revelou tempos de partida muito curtos (cerca de 20 dias), em todas as condições testadas, sendo atingidas remoções de DQO próximas de 70%. Além disso, o tipo de impelidor exerceu grande influência na qualidade final do efluente, fato este claramente constatado quando as frações de DQO foram consideradas separadamente (filtrada e suspensa). De acordo com os resultados obtidos na etapa 3, o tamanho da biopartícula teve influência decisiva no desempenho do sistema, nas condições testadas. As velocidades de dissolução foram aparentemente influenciadas pelo empacotamento do leito de espuma, enquanto que o consumo da fração de DQO correspondente às amostras filtradas foi provavelmente influenciado por fatores mais complexos. Finalmente, o teste realizado com resíduo diluído de suinocultura demonstrou que a operação do reator em estudo para o tratamento deste tipo de água residuária é possível. Os dados operacionais mostraram que o reator permaneceu estável durante o período testado. A agitação mecânica provou ser eficiente para melhorar a degradação da DQO suspensa, um dos maiores problemas no tratamento deste tipo de água residuária. Sendo assim, de acordo com os dados experimentais obtidos ao longo do trabalho, pode-se afirmar que a agitação em reatores em batelada mostrou-se importante não somente para proporcionar boas condições de mistura ou melhorar a transferência de massa na fase líquida, mas também para melhorar a solubilização da matéria orgânica particulada, melhorando as velocidades de consumo de matéria orgânica.

Enrichment of nitrifying microbial communities from shrimp farms and commercial inocula

Nitrifying bacteria were selected from shrimp farm water and sediment (natural seed) in Thailand and from commercial seed cultures. The microbial consortia from each source giving the best ammonia removal during batch culture pre-enrichments were used as inocula for two sequencing batch reactors (SBRs). Nitrifiers were cultivated in the SBRs with 100 mg NH4-N/I and artificial wastewater containing 25 ppt salinity. The two SBRs were operated at a 7 d hydraulic retention time (HRT) for 77 d after which the HRT was reduced to 3.5 d. The amounts of ammonia removed from the influent by microorganisms sourced from the natural seed were 85% and 92% for the 7 d HIRT and the 3.5 d HRT, respectively. The ammonia removals of microbial consortia from the commercial seed were 71% and 83% for these HRTs respectively. The quantity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) was determined in the SBRs using the most probable number (MPN) technique. Both AOB and NOB increased in number over the long-term operation of both SBRs. According to quantitative fluorescence in situ hybridisation (FISH) probing, AOB from the natural seed and commercial seed comprised 21 +/- 2% and 30 +/- 2%, respectively of all bacteria. NOB could not be detected with currently-reported FISH probes, suggesting that novel NOB were enriched from both sources. Taken collectively, the results from this study provide an indication that the nitrifiers from shrimp farm sources are more effective at ammonia removal than those from commercial seed cultures.

Comparison of acetate and propionate uptake by polyphosphate accumulating organisms and glycogen accumulating organisms

Enhanced biological phosphorus removal (EBPR) performance is directly affected by the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigates the effects of carbon source on PAO and GAO metabolism. Enriched PAO and GAO cultures were tested with the two most commonly found volatile fatty acids (VFAs) in wastewater systems, acetate and propionate. Four sequencing batch reactors (SBRs) were operated under similar conditions and influent compositions with either acetate or propionate as the sole carbon source. The stimulus for selection of the PAO and GAO phenotypes was provided only through variation of the phosphorus concentration in the feed. The abundance of PAOs and GAOs was quantified using fluorescence in situ hybridisation (FISH). In the acetate fed PAO and GAO reactors, Candidatus Accumulibacter phosphatis (a known PAO) and Candidatus Competibacter phosphatis (a known GAO) were present in abundance. A novel GAO, likely belonging to the group of Alphaproteobacteria, was found to dominate the propionate fed GAO reactor. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to take up acetate and propionate. PAOs enriched with acetate as the sole carbon source were immediately able to take up propionate, likely at a similar rate as acetate. However, an enrichment of GAOs with acetate as the sole carbon source took up propionate at a much slower rate (only about 5% of the rate of acetate uptake on a COD basis) during a short-term switch in carbon source. A GAO enrichment with propionate as the sole carbon source took up acetate at a rate that was less than half of the propionate uptake rate on a COD basis. These results, along with literature reports showing that PAOs fed with propionate (also dominated by Accumulibacter) can immediately switch to acetate, suggesting that PAOs are more adaptable to changes in carbon source as compared to GAOs. This study suggests that the PAO and GAO competition could be influenced in favour of PAOs through the provision of propionate in the feed or even by regularly switching the dominant VFA species in the wastewater. Further study is necessary in order to provide greater support for these hypotheses. (c) 2005 Wiley Periodicals, Inc.

Competition between polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems with acetate and propionate as carbon sources

Enhanced biological phosphorus removal (EBPR) is a widely used process for achieving phosphorus removal from wastewater. A potential reason for EBPR failure is the undesirable growth of glycogen accumulating organisms (GAOs), which can compete for carbon sources with the bacterial group responsible for phosphorus removal from wastewater: the polyphosphate accumulating organisms (PAOs). This study investigates the impact of carbon source on EBPR performance and the competition between PAOs and GAOs. Two sequencing batch reactors (SBRs) were operated during a 4-6 month period and fed with a media containing acetate or propionate, respectively, as the sole carbon source. It was found that the acetate fed SBR rarely achieved a high level of phosphorus removal, and that a large portion of the microbial community was comprised of Candidatus Competibacter phosphatis, a known GAO. The propionate fed SBR, however, achieved stable phosphorus removal throughout the study, apart from one brief disturbance. The bacterial community of the propionate fed SBR was dominated by Candidatus Accumulibacter phosphatis, a known PAO, and did not contain Competibacter In a separate experiment, another SBR was seeded with a mixture of PAOs and a group of alphaproteobacterial GAOs, both enriched with propionate as the sole carbon source. Stable EBPR was achieved and the PAO population increased while the GAOs appeared to be out-competed. The results of this paper suggest that propionate may provide PAOs with a selective advantage over GAOs in the PAO-GAO competition, particularly through the minimisation of Competibacter Propionate may be a more suitable substrate than acetate for enhancing phosphorus removal in EBPR systems. (c) 2005 Elsevier B.V. All rights reserved.

Impianti di depurazione delle acque: SBR

In questo elaborato è stato discusso il tema della depurazione delle acque reflue, in particolar modo il sistema SBR. L'SBR (Sequencing Batch Reactors) è un sistema di impianti di depurazione composto da una vasca di raccolta del liquame e una o più vasche in parallelo, i reattori, che consistono nel vero e proprio cure dell'impianto. Di fatto le fasi del processo depurativo restano immutate rispetto ad un impianto tradizionale, ma l'SBR introduce come nuovo parametro dimensionale il tempo. infatti, tutti i trattamenti depurativi sono svolti sequenzialmente nel reattore. Un programma gestisce le varie fasi del trattamento esclusivamente sulla base della loro durata (stabilita relativamente alla natura del refluo e quindi ai trattamenti da svolgere in maniera più o meno intensiva). Questa caratteristica rende l'intero impianto molto versatile nel caso di variazioni dei dati in ingresso. Per questo motivo gli impianti SBR sono ottimi per la piccola media impresa, in quanto sono facilmente adattabili alle variazioni stagionali di produzione. Sono inoltre possibili realizzazioni di impianti per il trattamento di ingenti portate, o elevate concentrazioni, ponendo più reattori in parallelo alimentati dalle stessa vasca di accumulo. In questo modo è possibile svolgere un maggiore numero di cicli depurativi al giorno e quindi rispettare i valori normativi in uscita dell'impianto. In conclusione questa tipologia d'impianto presenta notevoli vantaggi fra i quali anche quello di avere bassi costi operativi. La motivazione di ciò sta nel fatto che l'impianto lavora solo se ha effettivamente una portata, per cui se non vi è presenza di refluo il reattore non lavora e quindi non comporta costi.