Chlorella vulgaris e Porphyridium purpureum: duas microalgas com polissacarídeos e outros compostos com potencial de valorização

Chlorella vulgaris and Porphyridium purpureum are two eukaryotic microalgae that have been highlighted from the biotechnological point of view due to their capability of producing multiple interesting metabolites, such as pigments and polysaccharides, beyond protein and lipids. Chlorella vulgaris is green due to the high cholorophyll content, representing a valuable source of this food aditive. However, since chlorophylls are sensitive compounds, they degrade easily by various mechanisms during storage. Aiming to find food grade suitable conditions for isolated chlorophylls preservation, C. vulgaris chlorophylls were extracted with 96% ethanol to study the influence of temperature, light, alkaline conditions, and modified atmosphere on the stability of the color in ethanolic solutions. Under the conditions used, the color loss was mainly due to the intense light (photoperiod 24 h), followed by the hight temperature (60 °C). The loss of green color in the ethanolic solution with temperature followed the first-order kinetic, being more significant between 28 and 60 °C, with an activation energy of 74 kJ/mol. To lower temperatures C. vulgaris chlorophylls showed resistance to the degradation when preserved in ethanol solutions. The addition of NaOH and the inert argon-rich atmosphere did not exhibit a statistically positive effect on the color preservation. Thus, C. vulgaris ethanolic extract showed to be more stable when protected from light at room temperature or bellow. C. vulgaris ethanolic extract showed to be a suitable natural food additive to coloring food stuffs. As case of study, the cooked cold rice was colored to be used in sushi. The color remained stable up to 3 days of storage at 4 ºC, either in the presence or absence of light. Beyond the high content in chlorophylls, C. vulgaris is rich in starch and structural polysaccharides that could have also great potential to be valued as food ingredients. Therefore, to fulfill this hypothesis, starch digestibility was evaluated in raw and boiled biomass, showing 43% and 71% of glucose released, respectively. The low extraction yield of starch obtained with water (13%) allowed to infer protein hindrance. This was overcome by 1 M and 4 M KOH aqueous solutions that allowed to obtain an additional 51% of starch. The final residue left showed that only 16% of total starch remained unextracted. KOH solutions allowed also to obtain galactans composed by 1,3-, 1,6- and 1,3,6-linked galactose residues. These linkages were also observed in the polysaccharides recovered from the growth medium, showing similarity between the exopolysaccharides and those present in the cell wall. The extracellular polymeric material revealed in vitro immunostimulatory effect on B lymphocytes. Porphyridium purpureum, also recognized as Porphyridium cruentum, is a red saline microalga that have been aroused commercial interest to be used for feeding fish in aquaculture. This microalga is rich in proteins and floridean starch, having the ability to excrete high levels of sulfated polysaccharides (sEPS) into the growth medium. P. purpureum is easily cultivated and could change their growth rate and composition in response to environmental variations. Thus, the impact of growth medium salinity (18, 32, and 50 g/L NaCl) on P. purpureum cells growth, biomass composition and on the extracellular polysaccharides production yield and chemical structure were evaluated. A maximum growth estimated as 5.7×106 cells/mL was obtained for 32 g/L of NaCl, after 19 days of growth. Besides biomass composition was not greatly changed, the sEPS excretion yield reflected the effect of salinity, higher for 32 g/L of NaCl (90 mg/L). The growth medium salinity slightly changed the sulfation pattern of the glucuronoglucogalactoxylan, since sEPS produced from P. purpureum grown at lower salinity tend to be more sulfated in O-3 position of xylose and O-6 position of glucose, while at higher salinity the sEPS tend to be more sulfated in O-4 position of xylose and glucose. The sEPS produced at higher salinity also revealed higher content of linear 2-Gal, 3-Gal, and 4-Gal residues. In all samples, the most representative sugar residues were constituted by t-Xyl, t-Xyl4S, 3-Xyl, 4-Xyl, t-Glc, 3-Glc6S, t-Gal, and 2,3,4-Gal. The sEPS showed immunostimulatory effect on B lymphocytes in vitro, similarly to the effect also demonstrated by the C. vulgaris extracellular polysaccharides. P. purpureum sEPS could be produced at large scale at an outdoor 800 L-flat panel photobioreactor with higher excretion yield (144 mg/L), revealing the potential of industrial production and commercialization of sEPS. In conclusion, this PhD thesis significantly upgraded the knowledge about the biotechnological potential of C. vulgaris pigments and both starch and exopolysaccharides, as food ingredients, with the additional commercial advantage of the possibility of these metabolites’ co-extraction. Moreover, P. purpureum sEPS revealed biotechnological potential to be used in aquaculture to enhance humoral immunoactivity of fish.As microalgas eucarióticas Chlorella vulgaris e Porphyridium purpureum têm-se destacado do ponto de vista biotecnológico devido à capacidade de produzirem vários metabolitos com interesse comercial, como pigmentos e polissacarídeos, para além da proteína e dos lípidos. A Chlorella vulgaris é verde devido ao seu alto teor em clorofila, sendo uma fonte valiosa deste aditivo alimentar. No entanto, uma vez que as clorofilas são compostos bastante sensíveis, podem degradar-se facilmente por vários mecanismos quando armazenadas. Assim, com o objetivo de determinar as condições mais apropriadas para a preservação de clorofilas isoladas, as clorofilas da C. vulgaris foram extraídas com 96% de etanol para estudar a influência da temperatura, luz, alcalinidade e atmosfera modificada na estabilidade da cor nas soluções etanólicas. Nas condições avaliadas, a perda da cor verde deveu-se principalmente à ação da luz (com um fotoperíodo de 24 h), seguida da temperatura elevada (60 °C). A perda da cor verde das soluções etanólicas com o aumento da temperatura seguiu uma cinética de 1.ª ordem, sendo mais significativa entre os 28 e os 60 °C, apresentando uma energia de ativação de 74 kJ/mol. Para temperaturas mais baixas observou-se uma resistência das clorofilas à degradação quando preservadas em etanol. A adição de NaOH e a atmosfera inerte rica em árgon não apresentaram um efeito estatisticamente positivo na preservação da cor verde. Desta forma, o extrato etanólico de C. vulgaris foi mais estável quando preservado no escuro à temperatura ambiente ou a temperaturas mais baixas. Os extratos etanólicos da C. vulgaris mostraram ser um aditivo alimentar natural adequado para corar produtos alimentares de verde. Como caso de estudo, foi corado arroz cozido frio para ser usado em sushi. Esta cor manteve-se estável durante pelo menos 3 dias de armazenamento, tanto na presença como na ausência de luz. Para além do alto teor em clorofilas, a microalga C. vulgaris é rica em amido e polissacarídeos estruturais, que podem também ter elevado potencial de valorização enquanto ingredientes alimentares. Assim, de forma a testar esta hipótese, a digestibilidade do amido foi avaliada na biomassa crua e cozida de C. vulgaris, apresentando uma libertação de 43 e 71% de glucose, respetivamente. A baixa extratabilidade do amido obtido com água quente mostrou um impedimento devido à presença de proteína. Este impedimento de extração foi ultrapassado usando soluções aquosas de 1 M e de 4 M de KOH, permitindo obter mais 51% do total de amido da microalga. O resíduo final obtido após as extrações mostrou que apenas 16% do amido ficou por extrair. Para além disso, as soluções de KOH também permitiram obter galactanas contendo resíduos de galactose com ligações nos carbonos 1,3, 1,6 e 1,3,6. Estas ligações também foram observadas nos polissacarídeos recuperados do meio de cultura, mostrando uma similaridade entre os polissacarídeos extracelulares e os polissacarídeos presentes na parede celular. O material polimérico extracelular revelou um efeito imunoestimulador in vitro em linfócitos B. A microalga vermelha salina Porphyridium purpureum, também conhecida por Porphyridium cruentum, tem despertado interesse comercial para ser usada para alimentação de peixes em aquacultura. Esta microalga é rica em proteínas, amido florídeo e tem a capacidade de excretar elevados níveis de polissacarídeos sulfatados (sEPS) para o meio de cultura. A microalga P. purpureum é cultivada facilmente e pode alterar a sua taxa de crescimento e a sua composição em resposta a alterações ambientais. Neste contexto, foi avaliado o impacto da salinidade do meio de cultura (18, 32 e 50 g/L de NaCl) no crescimento celular da P. purpureum, assim como na composição da sua biomassa e no rendimento de produção e estrutura química dos seus polissacarídeos extracelulares. Um crescimento máximo estimado de 5.7×106 células/mL foi obtido no meio de cultura com uma salinidade de 32 g/L de NaCl, após 19 dias de crescimento. Apesar da composição da biomassa da microalga P. purpureum não mudar significativamente com o nível de salinidade do meio de cultura, o rendimento de excreção dos sEPS foi maior para a cultura com 32 g/L de NaCl (90 mg/L). Para além disso, a salinidade do meio de cultura também alterou ligeiramente o padrão de sulfatação das glucuronoglucogalactoxilanas, uma vez que os sEPS produzidos pela P. purpureum que cresceu com o menor nível de salinidade, tendem a ser mais sulfatados na posição O-3 da xilose e na posição O-6 da glucose, enquanto a salinidades superiores os sEPS tendem a ser mais sulfatados na posição O-4 da xilose e da glucose. Os sEPS produzidos com salinidades maiores também revelaram uma maior percentagem de resíduos lineares de 2-Gal, 3-Gal e 4-Gal. No entanto, em todas as amostras, os resíduos de açúcares mais representativos foram os t- Xyl, t-Xyl4S, 3-Xyl, 4-Xyl, t-Glc, 3-Glc6S, t-Gal e 2,3,4-Gal. Os sEPS mostraram um efeito imunoestimulador in vitro nos linfócitos B, à semelhança do efeito também demonstrado pelos polissacarídeos extracelulares da C. vulgaris. A produção destes sEPS pela microalga P. purpureum pode ser realizada a larga escala num fotobiorreator de placas planas verticais de 800 L com um bom rendimento de excreção (144 mg/L), comprovando a viabilidade destes sEPS para produção industrial e comercialização. Em conclusão, esta tese de doutoramento aumentou o conhecimento do potencial biotecnológico dos pigmentos e polissacarídeos da C. vulgaris como ingredientes alimentares, com a vantagem comercial adicional de poderem ser co-extraídos. Além disso, os polissacarídeos extracelulares sulfatados da microalga P. purpureum revelaram um grande potencial para serem usados na aquacultura, de forma a aumentar a atividade imunoestimuladora dos peixes.Programa Doutoral em Química Sustentáve

Filmes à base de quitosana enriquecidos com extratos de bagaço de uva

Mestrado em Biotecnologia AlimentarOs filmes obtidos a partir de recursos naturais e renováveis têm sido alvo de inúmeros estudos nos últimos anos. Entre estes, os constituídos à base de quitosana, um polissacarídeo derivado da quitina que apresenta propriedades importantes para a aplicação na área alimentar, nomeadamente, atividade antioxidante e antimicrobiana, propriedades biofuncionais e biocompatíveis, biodegradabilidade, não toxicidade e capacidade de formar filmes com boas propriedades mecânicas. O bagaço de uva, constituído pelas grainhas, películas e engaços, é o principal subproduto da indústria vinícola. Este subproduto, que geralmente é descartado, é constituído por compostos de interesse como triacilglicerídeos, tocoferóis e polifenóis. O principal objetivo deste trabalho foi desenvolver filmes à base de quitosana com extratos do bagaço de uva incorporados, de forma a melhorar as suas propriedades antioxidantes e de insolubilidade, mantendo as propriedades mecânicas e antimicrobianas dos filmes. Neste estudo foram obtidas três frações do bagaço de uva: 1) o extrato obtido com água quente constituído por 72 % de polissacarídeos, 20 % de proteínas e 6 % de compostos fenólicos totais, 2) as ceras extraídas com clorofórmio e 3) o óleo, obtido por extração com n-hexano, constituído por cerca de 70 % de ácido linoleico (C18:2) e 18 % de ácido oleico (C18:1). Os três extratos foram incorporados na solução de quitosana (1,5 % m/v) e os filmes foram obtidos por evaporação do solvente. A solubilidade dos vários filmes foi determinada em água (pH=6,5) e numa solução hidro-alcoólica ácida (pH=3,5). Os filmes com os óleos incorporados na proporção de 1:5 (óleo:quitosana) obtiveram uma redução de 27 % de perda de massa na solução ácida em relação ao filme controlo constituído apenas por quitosana. Os restantes filmes não apresentaram diferenças significativas de solubilidade em relação ao filme de quitosana. A atividade antioxidante dos filmes foi determinada por diferentes métodos (ABTS, DPPH, FRAP e pelo método do poder redutor). Os filmes de quitosana com polissacarídeos, ceras e óleo demonstraram uma atividade antioxidante que pode ser até 3 vezes superior, dependendo do método, relativamente ao controlo, demonstrando a importância da incorporação destes extratos nos filmes. Os ensaios mecânicos permitiram demonstrar que a incorporação dos vários extratos não prejudica as características mecânicas dos filmes de quitosana. Os filmes de quitosana e quitosana com óleo incorporado demonstraram atividade antimicrobiana de fungos no pão e de bactérias no sumo de maçã. Em conclusão, os filmes de quitosana produzidos por incorporação dos extratos obtidos do bagaço de uva, principalmente o das ceras e do óleo, parecem ser promissores para futuras aplicações alimentares.The study of the films obtained from natural and renewable sources have greatly increased in recent years. From these, those composed by chitosan, a polysaccharide derived from chitin which has important properties for food industry such as antioxidant, antimicrobial, biocompatible, biofunctional, biodegradability, non-toxicity and the great ability to form films with good mechanical properties. The grape pomace, which includes seeds, skins and stems, is an important by-product of winemaking industry. This wine by-product is usually discarded thought containing valuable compounds such as triglycerides, tocopherols and polyphenols, which could have application in the food industry. The aim of this work was to develop chitosan-based films with incorporation of grape pomace extracts in order to improve their insolubility and antioxidant properties, keeping their mechanical and antimicrobial properties. In this study, three fractions were obtained from grape pomace: 1) hot water extract consisting of 72 % polysaccharides, 20 % proteins and 6 % total phenolic compounds, 2) waxes, extracted with chloroform and 3) oil, obtained with n-hexane, comprising about 70 % linoleic acid (C18:2) and 18 % oleic acid (C18:1). These three extracts were added to a chitosan solution (1.5% w / v) and the films were obtained by solvent casting. The solubility of the chitosan-based films was determined in water (pH=6,5) and in an acidic hydro-alcoholic solution (pH=3,5). The chitosan-based films with incorporation of the oil in a ratio of 1:5 (oil:chitosan) showed a decrease of 27 % of weight loss in acidic solution comparing with control chitosan films. The remaining films did not show significant differences comparing with the solubility of the control film. The antioxidant activity of the chitosan-based films was determined by four different methods (ABTS, DPPH, FRAP and the reducing power essay). The chitosan-based films with polysaccharides, wax and oil demonstrated an antioxidant activity that can be up to 3 times higher, relative to the control, depending on the method of analysis. These results demonstrated the importance of incorporating these extracts in the chitosan films. Mechanical tests showed that the incorporation of the three extracts in chitosan films did not affect significantly the mechanical properties. Also, the chitosan-based films demonstrated antimicrobial activity against fungi on bread and bacteria in apple juice. In conclusion, the chitosan-based films prepared by incorporation of extracts obtained from grape pomace, mainly waxes and oil, seem to be promising for food applications

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

Unraveling who is who in methanogenic oil degradation

2015 Gulf of Mexico Oil Spill and Ecosystem ConferenceMethanogenesis from hydrocarbons is a potentially important component of attenuation in water and sediments impacted by oil spills. The largest fraction of crude oil consists of aliphatic hydrocarbons (AHC). Current knowledge on key microorganisms degrading alkenes is scarce and is a central question addressed in our research. A methanogenic hexadecene (Hxd)-degrading consortium was obtained from laboratory microcosms inoculated with anaerobic granular sludge, and characterized by 16S rRNA gene amplification, cloning and sequencing. We have learned by community analysis that the present bacteria belong mainly to Syntrophaceae and Synergistaceae families. A Syntrophus-like microorganism (96% similarity at genera level) is possibly involved in Hxd degradation. Known methanogens utilizing acetate and H2/CO2 were identified, namely Methanosaeta-, Methanobacterium- and Methanolinea-related microorganisms, and were likely the syntrophic partners in Hxd degradation. With these results we find hints for similar pathways involved in alkenes and alkanes biodegradation. For alkanes, complete degradation to methane can occur through syntrophic interactions between bacteria and methanogens. This is the first time that an alkene-degrading methanogenic mixed community is characterized. Novel microorganisms involved in AHC degradation could be identified. This information is useful for understanding who is doing what, and at what rate. It can be used for innovative biotechnological solutions for deep contaminated sites clean-up.info:eu-repo/semantics/publishedVersio

Toxicity of long chain fatty acids towards acetate conversion by Methanosaeta concilii and Methanosarcina mazei

Long-chain fatty acids (LCFA) can inhibit methane production by methanogenic archaea. The effect of oleate and palmitate on pure cultures of Methanosaeta concilii and Methanosarcina mazei was assessed by comparing methane production rates from acetate before and after LCFA addition. For both methanogens, a sharp decrease in methane production (> 50%) was observed at 0.5 mmol L1 oleate, and no methane was formed at concentrations higher than 2 mmol L1 oleate. Palmitate was less inhibitory than oleate, and M. concilii was more tolerant to palmitate than M. mazei, with 2 mmol L1 palmitate causing 11% and 64% methanogenic inhibition respectively. This study indicates that M. concilii and M. mazei tolerate LCFA concentrations similar to those previously described for hydrogenotrophic methanogens. In particular, the robustness of M. concilii might contribute to the observed prevalence of Methanosaeta species in anaerobic bioreactors used to treat LCFA-rich wastewater.The authors thank the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement no. 323009 and the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and Project RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462)

Effect of long-chain fatty acids (LCFA) on the prevalence and viability of hydrogenotrophic methanogens

Anaerobic degradation of long-chain fatty acids (LCFA) is essential for efficient biogas production from complex lipid-containing wastewaters. Methanogens play a key role in this process, but the general idea is that LCFA exert a toxic effect towards these microorganisms that impairs good methane recovery. In this work, the effect of saturated (palmitate, C16:0) and unsaturated (oleate, C18:1) LCFA towards hydrogenotrophic methanogens was studied in batch enrichments and in pure cultures. Methanospirillum hungatei and Methanobacterium formicicum were added to oleate- and palmitate-degrading enrichments, cultures OM and PM, and their prevalence was subsequently monitored by PCR-DGGE. M. formicicum grew in both OM and PM cultures, while M. hungatei only prevailed in PM culture. Viability tests using live/dead staining further confirmed that M. hungatei is more sensitive to oleate than M. formicicum. The percentage of damaged cells, caused by the exposure to 0.5 mM of oleate, was higher in M. hungatei cultures (99 ± 1 %) than in M. formicicum cultures (53 ± 10 %). These results suggest that oleate is more inhibitory to methanogens than palmitate, although methane production was not completely inhibited with either LCFA

Integrating metabolomics and targeted gene expression to uncover potential biomarkers of fungal/oomycetes-associated disease susceptibility in grapevine

Vitis vinifera, one of the most cultivated fruit crops, is susceptible to several diseases particularly caused by fungus and oomycete pathogens. In contrast, other Vitis species (American, Asian) display different degrees of tolerance/resistance to these pathogens, being widely used in breeding programs to introgress resistance traits in elite V. vinifera cultivars. Secondary metabolites are important players in plant defence responses. Therefore, the characterization of the metabolic profiles associated with disease resistance and susceptibility traits in grapevine is a promising approach to identify trait-related biomarkers. In this work, the leaf metabolic composition of eleven Vitis genotypes was analysed using an untargeted metabolomics approach. A total of 190 putative metabolites were found to discriminate resistant/partial resistant from susceptible genotypes. The biological relevance of discriminative compounds was assessed by pathway analysis. Several compounds were selected as promising biomarkers and the expression of genes coding for enzymes associated with their metabolic pathways was analysed. Reference genes for these grapevine genotypes were established for normalisation of candidate gene expression. The leucoanthocyanidin reductase 2 gene (LAR2) presented a significant increase of expression in susceptible genotypes, in accordance with catechin accumulation in this analysis group. Up to our knowledge this is the first time that metabolic constitutive biomarkers are proposed, opening new insights into plant selection on breeding programsinfo:eu-repo/semantics/publishedVersio

Long-chain fatty acids degradation by desulfomonile species and proposal of candidatus Desulfomonile palmitatoxidans

The datasets generated for this study can be found in the European Nucleotide Archive (ENA)–LS453291 (https://www.ebi.ac.uk/ena/browser/view/LS453291), PRJEB26656 (https://www.ebi.ac.uk/ena/browser/view/PRJEB26656), PRJEB35900 (https://www.ebi.ac.uk/ena/browser/view/PRJEB35900).Microbial communities with the ability to convert long-chain fatty acids (LCFA) coupled to sulfate reduction can be important in the removal of these compounds from wastewater. In this work, an enrichment culture, able to oxidize the long-chain fatty acid palmitate (C16:0) coupled to sulfate reduction, was obtained from anaerobic granular sludge. Microscopic analysis of this culture, designated HP culture, revealed that it was mainly composed of one morphotype with a typical collar-like cell wall invagination, a distinct morphological feature of the Desulfomonile genus. 16S rRNA gene amplicon and metagenome-assembled genome (MAG) indeed confirmed that the abundant phylotype in HP culture belong to Desulfomonile genus [ca. 92% 16S rRNA gene sequences closely related to Desulfomonile spp.; and ca. 82% whole genome shotgun (WGS)]. Based on similar cell morphology and average nucleotide identity (ANI) (77%) between the Desulfomonile sp. in HP culture and the type strain Desulfomonile tiedjei strain DCB-1T, we propose a novel species designated as Candidatus Desulfomonile palmitatoxidans. This bacterium shares 94.3 and 93.6% 16S rRNA gene identity with Desulfomonile limimaris strain DCB-MT and D. tiedjei strain DCB-1T, respectively. Based on sequence abundance of Desulfomonile-morphotype in HP culture, its predominance in the microscopic observations, and presence of several genes coding for enzymes involved in LCFA degradation, the proposed species Ca. Desulfomonile palmitatoxidans most probably plays an important role in palmitate degradation in HP culture. Analysis of the growth of HP culture and D. tiedjei strain DCB-1T with short- (butyrate), medium- (caprylate) and long-chain fatty acids (palmitate, stearate, and oleate) showed that both cultures degraded all fatty acids coupled to sulfate reduction, except oleate that was only utilized by HP culture. In the absence of sulfate, neither HP culture, nor D. tiedjei strain DCB-1T degraded palmitate when incubated with Methanobacterium formicicum as a possible methanogenic syntrophic partner. Unlike D. tiedjei strain DCB-1T, Ca. Desulfomonile palmitatoxidans lacks reductive dehalogenase genes in its genome, and HP culture was not able to grow by organohalide respiration. An emended description of the genus Desulfomonile is proposed. Our study reveals an unrecognized LCFA degradation feature of the Desulfomonile genus.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of project MORE (PTDC/AAG-TEC/3500/2014; POCI-01-0145-FEDER016575), and 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

Effect of sub-stoichiometric Fe(III) amounts on LCFA degradation by methanogenic communities

Long-chain fatty acids (LCFA) are common contaminants in municipal and industrial wastewater that can be converted anaerobically to methane. A low hydrogen partial pressure is required for LCFA degradation by anaerobic bacteria, requiring the establishment of syntrophic relationships with hydrogenotrophic methanogens. However, high LCFA loads can inhibit methanogens, hindering biodegradation. Because it has been suggested that anaerobic degradation of these compounds may be enhanced by the presence of alternative electron acceptors, such as iron, we investigated the effect of sub-stoichiometric amounts of Fe(III) on oleate (C18:1 LCFA) degradation by suspended and granular methanogenic sludge. Fe(III) accelerated oleate biodegradation and hydrogenotrophic methanogenesis in the assays with suspended sludge, with H2-consuming methanogens coexisting with iron-reducing bacteria. On the other hand, acetoclastic methanogenesis was delayed by Fe(III). These effects were less evident with granular sludge, possibly due to its higher initial methanogenic activity relative to suspended sludge. Enrichments with close-to-stoichiometric amounts of Fe(III) resulted in a microbial community mainly composed of Geobacter, Syntrophomonas, and Methanobacterium genera, with relative abundances of 83–89%, 3–6%, and 0.2–10%, respectively. In these enrichments, oleate was biodegraded to acetate and coupled to iron-reduction and methane production, revealing novel microbial interactions between syntrophic LCFA-degrading bacteria, iron-reducing bacteria, and methanogens.Portuguese Foundation for Science and Technology (FCT) under the scope of project MORE (POCI-01-0145-FEDER-016575), 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. The authors also acknowledge the financial support of FCT and European Social Fund through the grants attributed to S.A. Silva (SFRH/BD/122623/2016), A.L. Arantes (PD/BD/128030/2016), and J.C. Sequeira (SFRH/BD/147271/2019)info:eu-repo/semantics/publishedVersio