Intensification of methane production from waste frying oil in a biogas-lift bioreactor

"Available online 31 December 2020"Biogas upgrading from anaerobic digestion of waste frying oils (WFO) was accessed in this study. For that, two bioreactors (Rb-biogas-lift bioreactor with gas and liquid recirculation, Rc-control reactor with liquid recirculation) were fed three times per week, with a mixture of WFO, glycerol and volatile fatty acids (VFA). Rb produced 1.4 times more biogas with higher methane content (79%) than Rc (67%). Higher relative abundance of hydrogenotrophic methanogens (34%-39%) was observed in Rb, when compared to Rc (16%-21%). The relative abundance of Sprochaetia class, which includes some homoacetogens/syntrophic acetate oxidizing genera, was also higher in Rb. This work shows that biogas recirculation applied in the biogas-lift bioreactor facilitated WFO degradation, most probably due to the selective enrichment of hydrogenotrophic methanogens. Recirculation of CO2 present in the biogas and reverse homoacetogenesis (i.e. syntrophic acetate oxidation) seem to be the main factors involved in the stimulation of hydrogenotrophic methanogenesis.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

Hexadecane toxicity towards pure cultures of methanogens

Petroleum industry generates large volumes of hydrocarbon-containing wastewater, that may be treated and valorized by anaerobic conversion to methane. This process is performed by complex microbial communities and is only thermodynamically feasible at low hydrogen partial pressure, which is generally accomplished by the activity of hydrogenotrophic methanogens. However, alkanes, polyaromatic hydrocarbons and BTEX were shown to inhibit methanogenesis in mixed microbial cultures. This may be due to a direct inhibition of the methanogens, or may result from indirect inhibition, by disrupting the microbial relationships in the complex communities. To get more insights on this topic, the toxicity of aliphatic hydrocarbons towards pure cultures of hydrogenotrophic methanogens was assessed in this work. Aliphatic hydrocarbons represent the largest fraction of crude oil or petroleum-derived products, and hexadecane (HC) was chosen as model compound. Methane production from H2/CO2 (80:20%, 1.7x105 Pa) by Methanobacterium formicicum and Methanospirillum hungatei was measured in the presence of increasing HC concentrations (1, 5, 15 and 30 mM), and was compared with the controls without HC. For both methanogens, the methane production rate was significantly lower (p30 mM could be estimated for M. formicicum and M. hungatei, respectively. Therefore, M. hungatei is more tolerant to the presence of HC than M. formicicum, possibly due to the differences in cell wall structure and membrane lipid composition of the two species. Moreover, the relatively high IC50 values obtained are most likely related with the low HC solubility. Considering the typical range of hydrocarbon concentrations in wastewater from the petroleum industry, toxic effects from aliphatic hydrocarbons towards hydrogenotrophic methanogens will not be expected to occur during the anaerobic treatment of these type of wastewater.This study was supported by FCT under the scope of project MORE (PTDC/AAGTEC/3500/2014; POCI-01-0145-FEDER-016575), of the strategic funding of UID/BIO/04469/2019 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

Ciprofloxacin removal catalysed by conductive carbon materials

Current wastewater treatment technologies are not effective in the removal of pharmaceuticals. In anaerobic bioreactors, the electrons produced during the oxidation of organic matter can potentially be used for the biological reduction of pharmaceuticals. However, these reactions occur generally at a slow rate, due to electron transfer limitations, and might be accelerated through redox mediators (RM). Carbon nanomaterials (CM) have been effective RM in the biological reduction of other pollutants. For instance, CNT@2%Fe were found to increase 76-fold the biological reduction of Acid Orange 10. The magnetic properties of those composites allow their easier recover after the process by using a magnetic field. In this study, CNT and CNT@2%Fe were studied in the anaerobic removal of Ciprofloxacin (CIP). Furthermore, the potential contribution of adsorption and biodegradation processes for CIP removal was assessed. Toxicity assessment is highly important as it is desired that the products formed after the process are not more toxic than the initial compound. Moreover, the evaluation of the possible contribution of nanomaterials used in the process for the final toxic effect of threated solution, is crucial. In this sense, the detoxification of the treated solutions was evaluated towards Vibrio fischeri.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 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

Oleate degradation in a continuous microaerophilic bioreactor by a syntrophic co-culture together with facultative anaerobic bacteria

Introduction: In high-rate bioreactors, to degrade long-chain fatty acids (LCFA), syntrophic bacteria convert LCFA to acetate and hydrogen/formate, and hydrogenotrophic methanogens keep the H2/formate at low levels, which is essential to allow the continuous LCFA degradation. However, syntrophic bacteria are usually detected in low numbers and facultative anaerobic bacteria (FAB) were shown to be relatively more abundant, although their role is not yet clear. This work aims to study the microbial relationships between syntrophic LCFA-degrading bacteria, methanogens and FAB in oleate degradation, under anaerobic and microaerophilic conditions. Methodology: A synthetic microbial consortium composed by Syntrophomonas zehnderi (a syntrophic LCFA-degrading bacterium, Sz), Methanobacterium formicicum (a hydrogenotrophic methanogen, Mf) and two Pseudomonas spp. (FAB isolated from an oleate degrading bioreactor, I1 and I2) was studied in a continuous bioreactor fed with oleate, under strict anaerobic and microaerophilic conditions. During the first 112 days, the bioreactor was operated with the syntrophic co-culture (Sz+Mf) under strict anaerobic conditions. After, the two Pseudomonas isolates (I1+I2) were added to the bioreactor and microaerophilic conditions were provided by allowing the entrance of low oxygen amounts through continuous feeding from a tank containing oxygen in the headspace. The bioreactor operation was divided in 10 different periods that differ in oleate concentration in the feed (0.25 to 4.67 mmol L-1), hydraulic retention time (19 to 10 days), and oxygen concentration in the feeding tank headspace (21%, 10% and 5%). In parallel, I1 and I2 were characterized in batch assays for their capability to degrade oleate or acetate under aerobic conditions, as well as formate and hydrogen under microaerophilic conditions (5% O2). Results: In the continuous microaerophilic bioreactor, oleate was degraded by the synthetic community composed of Sz+Mf+I1+I2. Acetate was the main product and methane was detected only in low amounts. The highest oleate conversion to acetate was achieved at 5% O2. Our data indicate that the production of acetate from oleate, under microaerophilic conditions, was performed largely by S. zehnderi, and that Pseudomonas isolates were potentially alternative syntrophic partners. Indeed, additional batch tests support this suggestion, as they confirmed that Pseudomonas isolates (I1+I2) were able to consume formate and hydrogen under microaerophilic conditions. Conclusions: Our results suggest that FAB, particularly Pseudomonas spp., besides protecting the strict anaerobic community from oxygen toxicity, may also act as alternative hydrogen/formate scavengers for syntrophic LCFA-degrading bacteria.info:eu-repo/semantics/publishedVersio

Facultative anaerobic bacteria: key players in syntrophic fatty acids degradation under microaerophilic conditions

Anaerobic digestion (AD) is a mature technology that contributes to the sustainable development through the production of energy and products, using microbes as key players. Oxygen, formerly thought as the nemesis of AD, has been shown to benefit the AD processes when provided in vestigial doses [1]. The beneficial effects of micro-aeration have been attributed to the increased activity of facultative anaerobic bacteria (FAB). Besides being involved in fermentation and acidogenesis, FAB have been referred to act as a protective shield against the damaging effects of oxidative stress to the strict anaerobic communities [2]. However, their role in the syntrophic degradation of fatty acids is not clear. In this work, the relationship between syntrophic bacteria (Syntrophomonas wolfei (Sw) and S. zehnderi (Sz)), methanogens (Methanospirillum hungatei (Mh) and Methanobacterium formicicum (Mf)) and FAB (Pseudomonas strains (Ps)) was investigated, during the degradation of short (C4, butyrate), medium (C8, octanoate) and long (C16, hexadecanoate). The syntrophic co-cultures were pre-grown and the Pseudomonas spp. were further added, along with each substrate, over a range of O2 concentrations (0-2 % v/v). A second transfer was performed, exposing each of the cultures to O2 concentrations between 0-2% (v/v). In the presence of O2 (even at the lower concentrations) the activity of the syntrophic cocultures sharply decreased or even disappeared. However, in the presence of Pseudomonas, methane production occurred, reaching the theoretically expected at days 3, 8 and 28 under 0%, 1% and 2% O2, respectively. These results were obtained for C8 degradation in the presence of Sw+Mh+Ps. The same tendency was observed for C4 and C16 degradation with both consortia. Additionally, in the 2nd transfer, the cultures previously exposed to O2, maintained their activity being able of completely convert substrates to methane, under anoxic and microaerophilic conditions. These results show the essential role of Pseudomonas in the protection of syntrophic coculture activity allowing fatty acids degradation under microaerophilic conditions. Therefore, in real AD systems, where vestigial O2 can be detected, the presence of FAB may result in more stable, resilient, and functional syntrophic communities.FCT under UIDB/04469/2020 unit; and SFRH/BD/132845/2017 grantinfo:eu-repo/semantics/publishedVersio

Effect of carbon nanotubes on methane production in pure cultures of methanogens and in a syntrophic co-culture

ICBM-3 - 3rd International Conference on Biogas MicrobiologyConductive materials have been reported to enhance methane production by anaerobic microbial communities from a wide diversity of substrates 1 . The mechanisms involved are far from being fully understood. Many studies suggest that these materials facilitate direct interspecies electron transfer (DIET) between electrogenic bacteria and methanogens and that this mechanism is even dominant over interspecies hydrogen and formate transfer 2,3. The effect of conductive materials in pure cultures of methanogens or in co-cultures of typical fatty acid-degrading syntrophs with methanogenic partners was never studied. In this work, the effect of carbon nanotubes (CNT) on the activity of pure cultures of Methanobacterium formicicum, Methanospirillum hungatei, Methanosarcina mazei and Methanosaeta concilii, and in the co-culture of Syntrophomonas wolfei and Methanospirillum hungatei was evaluated. The results showed that CNT affect methane production by methanogens. Initial methane production rate (MPR) increased 17 and 6 times when M. formicicum and M. hungatei were incubated with 5g·L-1 CNT, respectively. M. mazei and M. concilii‘ activities were higher when exposed to CNT concentrations of 0.1 to 1g·L-1 , but lower with 5g·L-1 . Increasing CNT concentrations resulted in more negative redox potentials, which correlated with the increased methanogenic activity. Remarkably, in the absence of a reducing agent, but in the presence of CNT, the MPR was higher than in incubations with reducing agent, while no growth was observed without reducing agent and without CNT. MPR from butyrate increased 1.5 fold in the presence of CNT (5g.L-1 ), showing a positive effect of CNT on the syntrophic coculture. Indications of DIET by the presence CNT were not obtained. Rather, CNT directly affects the activity of methanogens, which creates new opportunities to improve methane production from waste and wastewater in anaerobic digesters.info:eu-repo/semantics/publishedVersio

Detoxification of ciprofloxacin in an anaerobic bioprocess supplemented with magnetic carbon nanotubes: contribution of adsorption and biodegradation mechanisms

In anaerobic bioreactors, the electrons produced during the oxidation of organic matter can potentially be used for the biological reduction of pharmaceuticals in wastewaters. Common electron transfer limitations benefit from the acceleration of reactions through utilization of redox mediators (RM). This work explores the potential of carbon nanomaterials (CNM) as RM on the anaerobic removal of ciprofloxacin (CIP). Pristine and tailored carbon nanotubes (CNT) were first tested for chemical reduction of CIP, and pristine CNT was found as the best material, so it was further utilized in biological anaerobic assays with anaerobic granular sludge (GS). In addition, magnetic CNT were prepared and also tested in biological assays, as they are easier to be recovered and reused. In biological tests with CNM, approximately 99% CIP removal was achieved, and the reaction rates increased ?1.5-fold relatively to the control without CNM. In these experiments, CIP adsorption onto GS and CNM was above 90%. Despite, after applying three successive cycles of CIP addition, the catalytic properties of magnetic CNT were maintained while adsorption decreased to 29 ± 3.2%, as the result of CNM overload by CIP. The results suggest the combined occurrence of different mechanisms for CIP removal: adsorption on GS and/or CNM, and biological reduction or oxidation, which can be accelerated by the presence of CNM. After biological treatment with CNM, toxicity towards Vibrio fischeri was evaluated, resulting in ? 46% detoxification of CIP solution, showing the advantages of combining biological treatment with CNM for CIP removal.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. Ana Rita Silva holds an FCT grant SFRH/BD/131905/2017. Cátia S.N. Braga holds an FCT grant SFRH/BD/132003/2017. This work was also financially supported by: Base Funding-UIDB/50020/2020 of the Associate Laboratory LSRE LCM-funded by national funds through FCT/MCTES (PIDDAC). OSGPS acknowledges FCT fund ing under the Scientific Employment Stimulus-Institutional Call CEECINST/00049/2info:eu-repo/semantics/publishedVersio

What is the role of Pseudomonas sp. in the anaerobic digestion of long-chain fatty acids?

Palmitate accumulation in continuous bioreactors fed with oleate appears to be the cause for frequent reports of process failure during the anaerobic treatment of lipids or LCFA-rich wastewaters. In these reactors, oleate can be converted to palmitate by microorganisms other than syntrophic bacteria (facultative anaerobic bacteria), and do not depend on the presence of active hydrogenotrophic methanogens. To study non-syntrophic oleate conversion to palmitate, methanogens were selectively eliminated by applying low hydraulic retention time in a continuous stirred tank reactor (CSTR) fed with oleate. A small plug flow reactor (PFR) was installed in series with the CSTR to allow growth of some of the washed out microorganisms in the form of a biofilm. The high amount of palmitate in the biofilm samples indicated the presence of the key microorganisms on oleate conversion to palmitate. A sample of the PFR biofilm rich in palmitate, collected after 22 days of operation, was subjected to Illumina sequencing showing that Pseudomonas was the predominant (42%) genus present. Two Pseudomonas isolates obtained from the same biofilm were used to study the role of these microorganisms on oleate biotransformation. Two bioreactors were inoculated with isolates 1 (reactor RI1) and 2 (reactor RI2) and fed with oleate at an organic loading rate of 1 g L-1 d-1 (period I) and 4 g L-1 d-1 (period II) in chemical oxygen demand (COD). In period I, oleate concentration in COD varied between 0.258 g L-1 and 0.740 g L-1 in reactor RI1, and between 0.192 g L-1 and 0.642 g L-1 in reactor RI2. In period II oleate concentration in COD was close to 1.500 g L-1 in RI1 and 1.000 g L-1 in RI2. Apparently oleate is being used by Pseudomonas isolates, however no intermediate product was detected. Probably some intermediate metabolite is being produced and is not being quantified. This hypothesis is currently under study, and may bring some light on the puzzling bioconversion of oleate into palmitate.info:eu-repo/semantics/publishedVersio

Digestão anaeróbia

As comunidades microbianas anaeróbias são constituídas por microrganismos pertencentes aos domínios Bactéria e Arquea, que vivem em ambientes onde o oxigénio molecular está ausente. Estas comunidades desempenham um papel fundamental na decomposição da matéria orgânica, num processo designado por digestão anaeróbia. São geralmente utilizadas no tratamento anaeróbio de águas residuais e resíduos sólidos em biorreator, promovendo a recuperação de energia na forma de biogás, bem como na regulação dos ciclos biogeoquímicos e na manutenção/recuperação da qualidade dos ecossistemas.info:eu-repo/semantics/publishedVersio

Microaeration in anaerobic digestion systems: effect of low oxygen concentrations on methanogenic communities

In anaerobic digestion (AD) systems, the coordinated activity of different microbial groups leads to the conversion of complex organic matter into methane. The presence of oxygen may cause negative effects on these processes, by inhibiting the growth and activity of obligate anaerobes, namely methanogens. Nevertheless, the exposure to small amounts of oxygen (microaeration) was shown to improve the AD processes, mainly by enhancing the activity of facultative bacteria. These bacteria promote the hydrolysis and fermentation of the organic macromolecules into various intermediates, using oxygen as final electron acceptor, thus increasing the availability of substrates for syntrophic bacteria and methanogens. The effect of low oxygen concentrations towards these two microbial groups has been seldom studied and was investigated in this work. For that, anaerobic sludge was incubated in batch bottles with acetate, H2/CO2 or ethanol until reaching the exponential growth phase. Then, second substrate addition was performed and oxygen was added at increasing concentrations (0, 0.5, 1.0, 2.5 and 5%). Compared with the controls (0% O2), O2 exposure significantly decreased the substrate consumption and initial methane production rate (MPR) from H2/CO2 or acetate, at all the concentrations tested. At 0.5% O2, MRP from these two substrates was inhibited by 31±5% and 39 ±10%, respectively. Nevertheless, the assays amended with acetate were incubated over 30 hours, and activity was recovered in the assays that received the lower % O2. In the assays with ethanol, significant effects on ethanol uptake, acetate production and MPR were only observed at 2.5% and 5% O2. At 2.5%, MRP inhibition was 36±7%. The lower impact of O2 in these assays may be related to stimulation of facultative bacteria by the presence of ethanol, that enhanced O2 removal from the media, allowing the methanogenic community to maintain its activity.This study was supported by FCT under the scope of project MORE (PTDC/AAGTEC/3500/2014; POCI-01-0145-FEDER-016575), of the strategic funding of UID/BIO/04469/2019 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