Biodegradação anaeróbia do ácido oleico

Mestrado em Métodos Biomoleculares AvançadosO ácido oleico (C18:1) é um Ácido Gordo de Cadeia Longa (AGCL), considerado especialmente problemático em processos de digestão anaeróbia uma vez que exerce um efeito inibitório sobre os microrganismos e adsorve aos agregados microbianos causando a sua flutuação e consequente “washout” em reactores anaeróbios de alta carga. Nesta tese, após uma introdução geral onde é apresentado o estado da arte, descrevem-se três estudos sobre biodegradação anaeróbia de ácido oleico. No capítulo 2, foi estudada uma biomassa altamente carregada (5985 mg CQO.g SV-1). Avaliouse a sua capacidade de degradar oleato, em “batch”, em concentrações entre 100 e 1500 mg/l. As fases “lag” que precederam o início da produção de metano aumentaram com o aumento da concentração, até um periodo de 17 dias verificado para a concentração mais elevada. A recuperação da CQOmetano não excedeu os 50% nas concentrações mais elevadas de 1000 e 1500 mg/l. Após a mineralização do substrato associado à biomassa, e para as mesmas concentrações, obtiveram-se fases “lag” máximas de 3 dias e recuperações da CQO-metano na ordem dos 80%, evidenciando o aumento da capacidade da biomassa em degradar oleato. A actividade metanogénica específica em acetato e em H2/CO2 como substratos individuais aumentou 2,6 e 2 vezes respectivamente, após a mineralização dos substrato associado à biomassa, confirmando a reversibilidade do efeito inibitório dos AGCL. No capítulo 3 efectuou-se o estudo dos intermediários da degradação do ácido oleico em ensaios “batch”. As duas biomassas utilizadas foram recolhidas do mesmo digestor mas uma delas foi aclimatizada aos AGCL por contacto num reactor à escala laboratorial durante 50 dias,enquanto que a outra biomassa não contactou previamente com AGCL. Os intermediários da degradação foram analisados nas fases líquida e sólida durante 30 dias. De um modo geral, e para as duas biomassas, verificou-se adsorção do ácido oleico à fase sólida durante os primeiros 50 minutos, sendo depois convertido a metano. No caso da biomassa não aclimatizada, a taxa de degradação foi aproximadamente linear não se verificando acumulação apreciável de acetato durante o ensaio. No caso da biomassa aclimatizada, após um periodo de 10 dias foi observado um decréscimo acentuado da quantidade do oleato seguido de uma acumulação transiente de acetato que foi posteriormente convertido a metano. Foram verificadas fases “lag” antes de se iniciar a produção de metano nos ensaios com as biomassas aclimatizada e não aclimatizada, mas após o início da produção de metano, o tempo necessário para mineralizar 830 mg/l de oleato foi de 5 e 12 dias, respectivamente. No capítulo 4, as alterações da comunidade microbiana desenvolvida num reactor durante o tratamento de um efluente contendo oleato, através da análise do gene 16S rRNA pelas técnicas de PCR-DGGE, revelou que a composição da comunidade bacteriana foi mais afectada do que a comunidade de arquaea.Oleic Acid (C18:1) is an unsaturated Long Chain Fatty Acid (LCFA), described as especially problematic in anaerobic digestion processes due to its microbial inhibitory effect as well as because it adsorbs onto microbial aggregates causing flotation and washout in high-rate anaerobic reactors. In this thesis, after a general introduction referring the context and motivation and presenting the state of the art, three experiments are described about anaerobic biodegradation of oleic acid. In chapter 2, a sludge heavily loaded with 5985 mg COD.g VS-1 was studied in terms of its capacity to biodegrade oleic acid in batch assays, in concentrations ranging from 100 to 1500 mg/l. Lag phases before the onset of methane production increased with the oleic acid concentration, up to 17 days for 1500 mg/l and methane recovery did not exceed 50% for the highest concentrations tested (1000 and 1500 mg/l). After the mineralization of the biomass-associated substrate, and for the same concentration range, maximum lag phases of three days and methane recoveries up to 80% were obtained, evidencing an improving capacity of the sludge to biodegrade oleic acid. Also the specific methanogenic activity with acetate and H2/CO2 as individual substrates increased 2.6 and 2 times respectively, after the mineralization of the biomass associated substrate, confirming the reversibility of the inhibitory effect of LCFA. In chapter 3, the intermediates of oleic acid degradation were also studied in batch assays. Two different sludges were sampled from the same digester but afterwards one was acclimated to LCFA by contact in a lab-scale reactor during around 50 days and the other one was not acclimated. LCFA intermediates were analyzed in the liquid and in the solid phases along a 30 days experiment. In general, for both sludges, oleic acid adsorbed to the solid phase in the first 50 minutes and was converted to methane afterwards. In the non-acclimated sludge the degradation rate was approximately linear and there was not an appreciable accumulation of acetate during the course of the experiment. In the acclimated sludge, after an initial period of 10 days a sharp decrease on oleic acid quantity in the solid phase was observed followed by a significant, but transient accumulation of acetate that was afterwards converted to methane. Although both sludges exhibited a lag phase before the onset of methane production, the acclimated one was able to mineralize 830 mg/l of oleic acid in 5 days whereas the non-acclimated one took around 12 days. The study of the anaerobic microbial community dynamics during discontinuous operation of a lab-scale reactor treating a synthetic dairy wastewater was also investigated by analysis of the 16S rRNA gene using the polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) method. The composition of the bacterial community, based on the analysis of the DGGE patterns, was more affected than the archaeal population

Anaerobic biodegradation of oleate by a highly loaded biomass before and after degrading the associated substrate

Oleic Acid (C18:1) is an unsaturated Long Chain Fatty Acid (LCFA), described as especially problematic in anaerobic digestion processes. In this work, a sludge heavily loaded with 5985 mgCOD.gVSˉ¹ was studied in terms of its capability to biodegrade oleic acid in batch assays, in concentrations ranging from 100 to 1500 mg.lˉ¹. Lag phases before the onset of methane production increased with the oleic acid concentration, up to 17 days for 1500 mg.lˉ¹and methane production did not exceed 50% recovery for the highest concentrations tested (1000 and 1500 mg.lˉ¹). After the mineralization of the biomass-associated substrate, and for the same concentration range, maximum lag phases of three days and methane recoveries up to 80% were obtained, evidencing an improving capacity of the sludge to biodegrade oleic acid. Also the specific methanogenic activity with acetate and H2/CO2 as individual substrates increased 3.6 and 2 times respectively, after the mineralization of the biomass associated substrate, confirming the reversibility of the inhibitory effect of LCFA, even when the sludge was heavily loaded with more than 5 g COD.gVSˉ¹.Fundação para a Ciência e a Tecnologia (FCT) - POCTI/CTA/46328/2002

Anaerobic digestion of oily wastewater as a valuable source of bioenergy

Lipids are a group of organic pollutants whose conversion into biogas has been considered very difficult. During the anaerobic treatment of lipid-rich wastewater this conversion generally decreases with the increase of the organic loading rate (OLR) applied, due to long chain fatty acids (LCFA) accumulation. To overcome this problem, correct equilibrium between LCFA accumulation and degradation should be assured [1, 2, 3], and discontinuous operation was proposed by Pereira et al. [1] as a strategy to achieve an efficient rate of methane production. Based on these results, Cavaleiro et al. [4] studied the treatment of an oleate-rich effluent in an anaerobic reactor operated in cycles, with continuous feeding phases and batch reaction phases. The results obtained showed that continuous treatment was possible, with efficient conversion of LCFA to methane, after acclimation of the microbial consortium through discontinuous operation. This work aimed the optimization of biogas production in a continuous reactor fed with an oleate-rich wastewater and inoculated with acclimated anaerobic sludge. Acclimation was performed through discontinuous operation in a lab scale reactor. During the experiment, the OLR applied was gradually increased from 5 to 31 kgCOD m-3 day-1, by decreasing the hydraulic retention time. From 5 to 21 kgCOD m-3 day-1 the increase of the OLR was followed by a fast increase of the methane production rate, towards an average value that was directly related with the OLR, showing that there was no inhibition of the anaerobic consortium. However, when the OLR was increased to 26 kgCOD m-3 day-1, methane production rate fluctuated around the same average value as in the previous period (16 kgCOD-CH4 m-3 day- 1). For 31 kgCOD m-3 day-1, methane production rate tended to decrease, possibly due to microbial inhibition or mass transfer limitations. From 21 to 31 kgCOD m-3 day-1 methane production rate was very instable, indicating that the OLR applied were higher than the optimum value for the microbial community. Maximum methane yield (100%) was obtained for the OLR of 12 kgCOD m-3 day-1, but continuous anaerobic treatment of an OLR as high as 21 kgCOD m-3 day-1 was possible with a methane yield of 72% and average COD removal efficiency of 99%. Nevertheless, 16 kgCOD-CH4 m-3 day-1 is likely the optimum OLR to be applied, in order to optimize methane production. Oily wastewater can be used as a valuable source of bioenergy by applying proper anaerobic digestion technology

Factors affecting polyhydroxyalkanoates biodegradation in soil

Polyhydroxyalkanoates (PHAs) are polymers with widespread applications, from medical devices to packaging. PHAs can be biodegradable in natural environments, such as soil, but the blend of PHA with other materials can change the polymer properties and consequently affect the biodegradation process. The composition of the microbial communities in soil also significantly affects the biodegradation, but other factors such as temperature, pH, and soil moisture, can also be determinant. These ecological and physic/chemical factors change in different seasons and in different soil layers. It is essential to know how these factors influence the PHAs biodegradation to understand the impact of PHAs in nature. This review compiles the results on PHA polymers and PHA blends biodegradation, with focus on laboratory tests. The main factors affecting PHA's biodegradation in soil, both in laboratory tests and in the environment are also discussed.Miguel Fernandes acknowledges the grant PD/BD/146195/2019 provided by the Portuguese Foundation for Science and Technology (FCT). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Iron compounds in anaerobic degradation of petroleum hydrocarbons: a review

Waste and wastewater containing hydrocarbons are produced worldwide by various oil-based industries, whose activities also contribute to the occurrence of oil spills throughout the globe, causing severe environmental contamination. Anaerobic microorganisms with the ability to biodegrade petroleum hydrocarbons are important in the treatment of contaminated matrices, both in situ in deep subsurfaces, or ex situ in bioreactors. In the latter, part of the energetic value of these compounds can be recovered in the form of biogas. Anaerobic degradation of petroleum hydrocarbons can be improved by various iron compounds, but different iron species exert distinct effects. For example, Fe(III) can be used as an electron acceptor in microbial hydrocarbon degradation, zero-valent iron can donate electrons for enhanced methanogenesis, and conductive iron oxides may facilitate electron transfers in methanogenic processes. Iron compounds can also act as hydrocarbon adsorbents, or be involved in secondary abiotic reactions, overall promoting hydrocarbon biodegradation. These multiple roles of iron are comprehensively reviewed in this paper and linked to key functional microorganisms involved in these processes, to the underlying mechanisms, and to the main influential factors. Recent research progress, future perspectives, and remaining challenges on the application of iron-assisted anaerobic hydrocarbon degradation are highlighted.This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of project MORE (POCI-01-0145-FEDER-016575) and of the strategic funding of UIDB/04469/2020 unit. It was also funded by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020, and by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte—BioEcoNorte project (NORTE-01-0145-FEDER-000070).info:eu-repo/semantics/publishedVersio

Improving analysis of meta-omics data with the MOSCA framework

Introduction: Meta-omics is an emergent field of research with many resources available in the form of databases and software. The information stored in databases is not always easily accessible, and software tools for meta-omics are often difficult to utilize. In this work, we present Meta-Omics Software for Community Analysis (MOSCA), a software framework that implements pipelines for the integrated analysis of metagenomics (MG), metatranscriptomics (MT) and metaproteomics (MP) data. This framework integrates tools allowing access to databases, handling of data and a complete workflow for meta-omics data analysis. Methodology and results: MOSCA was developed in Python 3, takes as input raw files obtained from Next-generation sequencing (in FastQ format), and from mass spectrometry (mass spectra in vendor or peak-picked formats), and integrates several tools for MG, MT and MP analysis. These tools are connected through their inputs/outputs by snakemake, in a fully automated workflow. MG analysis starts with preprocessing of sequencing reads, which automatically configures Trimmomatic to remove adapters and low-quality reads based on FastQC quality reports, and SortMeRNA for rRNA reads removal. Assembly can be performed with MetaSPAdes or Megahit and is followed by binning with MaxBin2 and CheckM for quality check. Genes are identified with FragGeneScan and are annotated with both UPIMAPI (homology-based annotation) and reCOGnizer (domain-based annotation), with reference to UniProt KB and eight databases included in the Conserved Domains Database, respectively. Bowtie2 is used to align reads to metagenomes. Protein identification and quantification can be performed with either SearchCLI coupled to PeptideShaker (performing peptide-to-spectrum matching and spectra count) or using MaxQuant (with quantification at the MS1 level). Differential gene expression analysis is performed with DESeq2, and heatmaps, volcano plots and PCA plots are generated. The expressed enzymes are plotted into hundreds of KEGG metabolic maps with the tool KEGGCharter, showing the metabolic functions that are differentially expressed and the taxonomic assignment. Tables, heatmaps and other representations obtained with MOSCA provide an interactive, accessible and comprehensive representation of the information obtained from MG, MT and MP analyses. Conclusions: MOSCA performs automatic analyses of MG, MT and MP datasets, integrating over 20 tools to obtain a comprehensive and easy to understand representation of microbial activity in different processes and conditions.info:eu-repo/semantics/publishedVersio

UPIMAPI, reCOGnizer and KEGGCharter: three tools for functional annotation

Omics technologies generate large datasets from which biological information must be extracted by using bioinformatics tools. Although web services provide easier to use interfaces, large datasets are difficult to handle. This is not a limitation of command-line tools and programmatic modules, but these may be challenging to use. In this work, three command-line tools were developed, aimed for speed and automation. The tools are available through Bioconda for Unix systems and were developed in Python 3, making use of multithreading/multiprocessing in computationally demanding steps. UPIMAPI integrates annotation with reference to the UniProt database with automatic retrieval of internal and cross-reference information from other databases (e.g., KEGG, BRENDA and RefSeq) through UniProts API, accessed with urllib package. The input is a FASTA file containing protein sequences, and the outputs are EXCEL or TSV files containing taxonomic, functional, and cross-reference information. reCOGnizer performs domain-based annotation of protein sequences with CDD, Pfam, NCBIfam, Protein Clusters, TIGRFAM, SMART, COG and KOG as reference databases, and obtains EC numbers and taxonomic assignments per domain identified. The results are outputted in TSV and EXCEL files. KEGGCharter is a command line implementation of KEGG Pathways mapping service, while also obtaining additional KOs and EC numbers, through the methods available in BioPython for accessing KEGGs API. KEGGCharter takes as input a table (TSV or EXCEL), containing either KEGG IDs, KOs or EC numbers. KEGGCharter represents identified KOs in metabolic maps and includes information on differential gene expression. When data from more than one organism is uploaded, KEGGCharter links function to taxonomic identification, which can be visualized in the maps. Differential expression of genes/proteins can be visualized in metabolic maps, by showing mini heatmaps. UPIMAPI and reCOGnizer are complementary tools, providing functional annotation based on protein sequencing homology and on identification of protein conserved domains, respectively. Both tools retrieve the IDs (KEGG IDs, EC numbers and KOs) necessary to run KEGGCharter. Together, these tools provide a complete characterization and visualization of results, facilitating the interpretation of omics experiments, and requiring minimal bioinformatics expertise.info:eu-repo/semantics/publishedVersio

Functional analysis of syntrophic LCFA-degrading microbial ecosystems

ICBM 2014 - 2nd International Conference on Biogas Microbiology[Excerpt] Introduction and Aim: Long-chain fatty acids (LCFA) are present in lipid rich wastewater and can be converted to methane anaerobically, coupling wastewater treatment to bioenergy production. Differences in the degradation of saturated and unsaturated long-chain fatty acids (LCFA) by anaerobic consortia are not completely understood. Previous studies showed a segregation on the microbial community composition when the same inoculum sludge was incubated with saturated- or with unsaturated-LCFA (Sousa et al., 2007), suggesting differences in their degradation pathways. In order to get more insights on this and aiming linking microbial community structure to function, a comparative shotgun metaproteomics study of a mesophilic anaerobic sludge incubated with saturated- and unsaturated-LCFA was conducted. Additionally, the metaproteome of a defined co-culture of Syntrophomonas zehnderi and Methanobacterium formicicum growing on saturated- and unsaturated-LCFA to methane was also analyzed. [...]The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013, the project “BioEnv - Biotechnology and Bioengineering for a sustainable world”, REF. NORTE-07-0124-FEDER-000048” co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER and the project FCOMP-01-0124-FEDER-014784, financed by the FEDER funds through the Operational Competitiveness Programme (COMPETE) and by national funds through the FCT.info:eu-repo/semantics/publishedVersio

Methane production and conductive materials: a critical review

Conductive materials (CM) have been extensively reported to enhance methane production in anaerobic digestion processes. The occurrence of direct interspecies electron transfer (DIET) in microbial communities, as an alternative or complementary to indirect electron transfer (via hydrogen or formate), is the main explanation given to justify the improvement of methane production. Not disregarding that DIET can be promoted in the presence of certain CM, it surely does not explain all the reported observations. In fact, in methanogenic environments DIET was only unequivocally demonstrated in cocultures of Geobacter metallireducens with Methanosaeta harundinacea or Methanosarcina barkeri and frequently Geobacter sp. are not detected in improved methane production driven systems. Furthermore, conductive carbon nanotubes were shown to accelerate the activity of methanogens growing in pure cultures, where DIET is not expected to occur, and hydrogenotrophic activity is ubiquitous in full-scale anaerobic digesters treating for example brewery wastewaters, indicating that interspecies hydrogen transfer is an important electron transfer mechanism in those systems. This paper presents an overview of the effect of several iron-based and carbon-based CM in bioengineered systems, focusing on the improvement in methane production and in microbial communities changes. Control assays, as fundamental elements to support major conclusions in reported experiments, are critically revised and discussed.This study was supported by the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement no. 323009 and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145FEDER-006684) and project PAC MultiBiorefinery SAICTPAC/0040/2015 (POCI-01-0145-FEDER-016403), and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. We acknowledge the fellowships awarded to Gilberto Martins (SFRH/BPD/80528/2011) and Luciana Pereira (SFRH/BPD/110235/2015) under the scope of the program POPH/ESF.info:eu-repo/semantics/publishedVersio

Anaerobic biological removal of pharmaceuticals: impact of these micropollutants towards different microbial groups in anaerobic communities

Pharmaceutical compounds are originated essentially from anthropogenic activities and end up in wastewater treatment plants (WWTP). Despite the low concentrations usually detected in wastewater (ranging from ng L-1 to g L-1), levels of mg L-1 have been detected in some countries. Moreover, in conventional WWTP these compounds are hardly degraded and tend to accumulate in sludge, being an environmental and public health problem. A possible treatment and valorization of contaminated sewage sludge is through anaerobic digestion, but for that purpose, the effect of these micropollutants on the activity of crucial microbial groups present in the anaerobic sludge (i.e., acetogenic and methanogenic microorganisms) must be assessed. In this work, the effect of ciprofloxacin (CIP), ibuprofen (IBP), diclofenac (DCF), and 17-ethinylestradiol (EE2), on the activity of acetogenic and methanogenic anaerobic communities was investigated1. The microorganisms respond dissimilarly to these micropollutants, at different concentrations (0.01-100 mg L-1), but in general they were more affected by CIP, followed by EE2, DCF and IBP. The specific methanogenic activity (SMA) was not affected in concentrations ranging from 0.01 to 0.1 mg L-1. However, acetoclastic methanogens were the most sensitive microorganisms, being affect by all the pharmaceuticals, at higher concentrations. The SMA of these microorganisms was inhibited 20% by 1 mg L-1 of CIP, and circa 50% with higher concentrations. Acetogenic bacteria were not affected by IBP at all the tested concentrations, but they were sensitive to CIP at concentrations above 1 mg L-1, and to DCF and EE2 at concentrations above 10 mg L-1. Instead, hydrogenotrophic methanogens were not affected by any concentration, indicating their lower sensitivity. It can be concluded that methanogenic communities were not severely affected by these pharmaceuticals. So, the application of anaerobic digestion for the treatment of wastewater and sewage sludge contaminated with pharmaceuticals seems promising. Indeed, another study showed that CIP can be removed by anaerobic sludge in the presence of carbon materials (99% removal), and treated wastewater was much less toxic than before the treatment (46% detoxification), as assessed with the standard bioassay using Vibrio fischeri.info:eu-repo/semantics/publishedVersio