Novel insights in dimethyl carbonate-based extraction of polyhydroxybutyrate (PHB)

Background: Plastic plays a crucial role in everyday life of human living, nevertheless it represents an undeniable source of land and water pollution. Polyhydroxybutyrate (PHB) is a bio-based and biodegradable polyester, which can be naturally produced by microorganisms capable of converting and accumulating carbon as intracellular granules. Hence, PHB-producing strains stand out as an alternative source to fossil-derived counterparts. However, the extraction strategy affects the recovery efficiency and the quality of PHB. In this study, PHB was produced by a genetically modified Escherichia coli strain and successively extracted using dimethyl carbonate (DMC) and ethanol as alternative solvent and polishing agent to chloroform and hexane. Eventually, a Life Cycle Assessment (LCA) study was performed for evaluating the environmental and health impact of using DMC. Results: Extraction yield and purity of PHB obtained via DMC, were quantified, and compared with those obtained via chloroform-based extraction. PHB yield values from DMC-based extraction were similar to or higher than those achieved by using chloroform (≥ 67%). To optimize the performance of extraction via DMC, different experimental conditions were tested, varying the biomass state (dry or wet) and the mixing time, in presence or in absence of a paper filter. Among 60, 90, 120 min, the mid-value allowed to achieve high extraction yield, both for dry and wet biomass. Physical and molecular dependence on the biomass state and solvent/antisolvent choice was established. The comparative LCA analysis promoted the application of DMC/ethanol rather than chloroform/hexane, as the best choice in terms of health prevention. However, an elevated impact score was achieved by DMC in the environmentallike categories in contrast with a minor contribution by its counterpart. Conclusion: The multifaceted exploration of DMC-based PHB extraction herein reported extends the knowledge of the variables affecting PHB purification process. This work offers novel and valuable insights into PHB extraction process, including environmental aspects not discussed so far. The findings of our research question the DMC as a green solvent, though also the choice of the antisolvent can influence the impact on the examined categories.This work has been funded by the Horizon 2020 EU Framework Programme: CELBICON project, Grant agreement number: 679050. Open access funding provided by PRIME project funded by the POR FESR 2014/2020 Programme, Asse I – Azione I.1b.2.2 Regione Piemonte, within the Piattaforma Tecnologica per la Bioeconomia.Publicad

Biological Aspects, Advancements and Techno-Economical Evaluation of Biological Methanation for the Recycling and Valorization of CO2

Nowadays, sustainable and renewable energy production is a global priority. Over the past decade, several Power-to-X (PtX) technologies have been proposed to store and convert the surplus of renewable energies into chemical bonds of chemicals produced by different processes. CO2 is a major contributor to climate change, yet it is also an undervalued source of carbon that could be recycled and represents an opportunity to generate renewable energy. In this context, PtX technologies would allow for CO2 valorization into renewable fuels while reducing greenhouse gas (GHG) emissions. With this work we want to provide an up-to-date overview of biomethanation as a PtX technology by considering the biological aspects and the main parameters affecting its application and scalability at an industrial level. Particular attention will be paid to the concept of CO2-streams valorization and to the integration of the process with renewable energies. Aspects related to new promising technologies such as in situ, ex situ, hybrid biomethanation and the concept of underground methanation will be discussed, also in connection with recent application cases. Furthermore, the technical and economic feasibility will be critically analyzed to highlight current options and limitations for implementing a sustainable process

“Innovative high pressure/high temperature, multi-sensing bioreactors system for microbial risk assessment in underground hydrogen storage”

This study addresses the microbial risks associated with Underground Hydrogen Storage (UHS), a critical component in the transition towards renewable energy systems, by employing an innovative multi-reactor system (Bio-xplorer) to simulate UHS conditions in two Italian reservoirs. The microbiological risk assessment (MRA) of Reservoir A and B was evaluated by subjecting them to gas mixtures of 10 % H2 and 90 % CH4, and 99 % H2 and 1 % CO2, respectively. In Reservoir A, the stability of pressure and temperature, the negligible optical density, and lack of microbial metabolites suggested a low risk of microbial activation. Molecular analyses confirmed the absence of sulphate- reducing bacteria (SRB) and limited growth of hydrogenotrophic methanogens (HM). Similarly, in Reservoir B, the absence of SRB and limited occurrence of HM indicated a low microbiological risk. Overall, the present work supports the safe and efficient implementation of UHS, a promising mitigation technique for climate change, using an innovative tool for MRA

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

From CO2 to CH4 via biological methanation

The leitmotif of this Ph.D. thesis is represented by carbon dioxide (CO2) recycling via biological production of methane (CH4). This work particularly focuses on the physiology of three hydrogenotrophic methanogens, Methanothermobacter marburgensis (M. marburgensis), Methanothermococcus okinawensis (M. okinawensis) and Methanococcus maripaludis S2 (M. maripaludis), that can be used as catalysts for biological methane production (BMP) process. This CO2 recycling method is challenging due to an inefficient transfer rate of molecular hydrogen (H2) from the gas phase to the liquid phase. Thus, the biocatalyst performance is limited by H2 availability in the liquid medium. However, several factors, as strain type and media requirements, operating conditions, and reactor design, can contribute to the success of CO2 conversion to CH4. Understanding the physiology of methanogens is a powerful tool for developing a scalable BMP process. Therefore, a novel study on the role of trace metals in pure cultures of M. okinawensis and M. marburgensis respectively is herein proposed. Experimental method of this study included an in silico analysis, closed batch, and fed-batch cultivations. In silico analysis revealed genomic differences among the transport systems and enzymes related to the methanogenesis pathway of these two methanogens. The importance of Fe as metal cofactor in methanogenesis emerged from the in silico analysis and it has been confirmed by the closed batch and fed-batch experiments. M. okinawensis responded to rising concentrations of trace element (TE) by increasing specific growth rate (µ, h-1) and volumetric productivity of methane (MER, mmolL-1h-1) during closed batch cultivation. Furthermore, M. okinawensis shown growth and CH4 in fed-batch cultivation. On the base of fed-batch cultures results, M. marburgensis was prioritized and applied for CO2-based BMP process optimization. It has been proposed a new feeding strategy based on exponential fed-batch cultivation where different medium-, TE- and sulphide dilution rates combinations, and different CO2/H2 inflow rates corresponded to a defined run. The specific setting of each run produced different responses from M. marburgensis. In this context, a MER of 476 mmol L-1 h-1 and µ of 0.69 h-1 were eventually achieved at highest H2/CO2 gassing rate and ratio. However, if these factors mitigate the limitation due to the H2 mass transfer on one side, they also reduce CH4 purity in the offgas on the other side. The combined effect of increasing TE dilution and H2/CO2 gassing rates positively affected the biomass and biomass concentration. Among trace elements, there are heavy metals whose toxicity is higher than others. Heavy metals can seriously affect the functionality of microorganisms, and therefore compromise their performances as biocatalysts of a bio-based process. Not only metals, but also organic compounds, such as carboxylic acids, can damage cells survival. Thus, the second experimental part of this thesis deals with inhibition studies on pure culture of M. maripaludis in closed batch cultivation. Despite the potential applications of M. maripaludis, the knowledge surrounding this strain runs out of lab-scale studies concerning the physiology and toxicology of heavy metals and VFAs. Therefore, M. maripaludis growth and productivity were tested by using copper (Cu), zinc (Zn), acetate (Ac) and propionic acid (Pr) as potential inhibitors of microbial activity. The culture was totally inhibited at concentration of 30, 70 and 100 mgL-1 of Cu and 0.7 and 1 gL-1 of Zn. However, M. maripaludis shows tolerance to 3, 7 and 10 mgL-1 of Cu with different extent. The addition of 0.3 gL-1 of Zn to the medium, rather promoted the biomass build-up of M. maripaludis and cancelled the effect of Cu when used together in the medium. In this study, it has been supported that the inhibition by Cu is due to a reduced or suppressed activity of the CODH/ACS complex producing acetyl-CoA intermediate. Acetyl-CoA is the precursor of many metabolic subsystems (e.g. lipid, amino acids, nucleotides pathways) and its alteration would interfere with them. While CODH/ACS activity is supported by CO2 and methanogenesis intermediate, the other way to produce acetyl-CoA is based on the acetate:CoA ligase. The relevance and the tolerance to rising concentrations of Ac and Pr was also investigated and quantified via HPLC analysis. Concentration of 5 and 10 mgL-1 of acetate did not inhibit nor growth neither productivity. Interestingly, the deprivation of acetate not only impacted on the growth rate but also on methanogenesis in M. maripaludis. In absence of Ac, the same concentrations of Pr caused a slow-down of the growth, while productivity was not touched. This study sheds light on the individual and combined impact of Cu, Zn, acetate and propionic acid on the metabolism of M. maripaludis. Furthermore, an attempt to define a possible mechanism which regulates specific acetate capture is provided in this study and the relevance of acetate:CoA ligase respect to CODH/ACS complex for acetyl-CoA synthesis is herein discussed. The information collected in this study are essential to improve the process efficiency of CO2 conversion to CH4 and extend the knowledge on the physiology of certain compounds. The tendency of these methanogens to adapt to adverse conditions, most of the time, offers the possibility to improve the engineering aspects of a limited process toward an unlimited one. Moreover, as a future activity, this thesis proposed the use of a 10-bar pressure bioreactor which has been projected in the frame of the Ph.D. research with a view to improving the success of biological CH4 production

The physiological effect of heavy metals and volatile fatty acids on Methanococcus maripaludis S2

Abstract Background Methanogenic archaea are of importance to the global C-cycle and to biological methane (CH4) production through anaerobic digestion and pure culture. Here, the individual and combined effects of copper (Cu), zinc (Zn), acetate, and propionate on the metabolism of the autotrophic, hydrogenotrophic methanogen Methanococcus maripaludis S2 were investigated. Cu, Zn, acetate, and propionate may interfere directly and indirectly with the acetyl-CoA synthesis and biological CH4 production. Thus, these compounds can compromise or improve the performance of M. maripaludis, an organism which can be applied as biocatalyst in the carbon dioxide (CO2)-based biological CH4 production (CO2-BMP) process or of methanogenic organisms applied in anaerobic digestion. Results Here, we show that Cu concentration of 1.9 µmol L−1 reduced growth of M. maripaludis, whereas 4.4 and 6.3 µmol L−1 of Cu even further retarded biomass production. However, 1.0 mmol L−1 of Zn enhanced growth, but at Zn concentrations > 2.4 mmol L−1 no growth could be observed. When both, Cu and Zn, were supplemented to the medium, growth and CH4 production could even be observed at the highest tested concentration of Cu (6.3 µmol L−1). Hence, it seems that the addition of 1 mmol L−1 of Zn enhanced the ability of M. maripaludis to counteract the toxic effect of Cu. The physiological effect to rising concentrations of acetate (12.2, 60.9, 121.9 mmol L−1) and/or propionate (10.3, 52.0, 104.1 mmol L−1) was also investigated. When instead of acetate 10.3 mmol L−1 propionate was provided in the growth medium, M. maripaludis could grow without reduction of the specific growth rate (µ) or the specific CH4 productivity (qCH4). A combination of inorganic and/or organic compounds resulted in an increase of µ and qCH4 for Zn/Cu and Zn/acetate beyond the values that were observed if only the individual concentrations of Zn, Cu, acetate were used. Conclusions Our study sheds light on the physiological effect of VFAs and heavy metals on M. maripaludis. Differently from µ and qCH4, MER was not influenced by the presence of these compounds. This indicated that each of these compounds directly interacted with the C-fixation machinery of M. maripaludis. Until now, the uptake of VFAs other than acetate was not considered to enhance growth and CH4 production of methanogens. The finding of propionate uptake by M. maripaludis is important for the interpretation of VFA cycling in anaerobic microenvironments. Due to the importance of methanogens in natural and artificial anaerobic environments, our results help to enhance the understanding the physiological and biotechnological importance with respect to anaerobic digestion, anaerobic wastewater treatment, and CO2-BMP. Finally, we propose a possible mechanism for acetate uptake into M. maripaludis supported by in silico analyses

Trace Elements of methanogens in an astrobiological context

Trace elements represent just a small part in the volume of a standard growth medium. These micronutrients are necessary for supporting the growth and metabolism of all life forms. The present study focuses on hydrogenotrophic methanogens. For these anaerobic microorganisms, trace elements are of particular importance due to their usage of the archaeal version of the Wood-Ljungdahl pathway. This pathway might be used by the last universal common ancestor in a hydrothermal vent setting. However, studies on the physiology of pure cultures of methanogens growing on H2/CO2 showed that the required optimal concentration of trace elements for microbial microorganisms is much higher than in the natural environments . The level of some trace elements in natural anoxic waters was even found to be below the concentration required for growth of methanogens in pure cultures. In this study, we investigate the effects of varying trace element concentrations on the growth of the two hydrogenotrophic methanogenic strains Methanothermobacter marburgensis and Methanothermococcus okinawensis. When varying the amount of trace elements the productivity and physiology of the two tested methanogens could be altered

Highlighting the Role of Archaea in Urban Mine Waste Exploitation and Valorisation

E-materials become e-waste once they have been discarded without the intent of reuse. Due to its rich content of metals, among which many are Critical Raw Materials (CRMs), e-waste can be considered an urban mine to exploit and valorise. Common metal refining is performed by energy-intensive processes frequently based on the use of fossil fuel. Bio-metallurgy is a promising alternative for e-waste valorisation based on biological routes of specialised microorganisms able to leach solid-containing metals. Because of the physiology of these microorganisms, microbial leaching can be economically feasible, besides being an environmentally sustainable process. Like Bacteria and Fungi, Archaea are also capable of metal leaching activity, though their potential is underestimated. Among them, the extremophiles are the most studied and applied in the field of metal recovery, while mesophilic species are less common but still of high interest. Here we provide the state of industrial application of bio-metallurgy and report on the state of the art of Archaea exploitation in metal recovery from e-waste. Moreover, we give a special highlight to methanogenic archaea, which are able to convert CO2 into methane in order to highlight the potential for the valorisation of CO2-rich industrial streams generated by key processes (i.e., anaerobic digestion, concrete, and steel production) in CH4 for gas grid distribution, while making metals content in e-waste available again as raw material

Highlighting the Role of Archaea in Urban Mine Waste Exploitation and Valorisation

E-materials become e-waste once they have been discarded without the intent of reuse. Due to its rich content of metals, among which many are Critical Raw Materials (CRMs), e-waste can be considered an urban mine to exploit and valorise. Common metal refining is performed by energy-intensive processes frequently based on the use of fossil fuel. Bio-metallurgy is a promising alternative for e-waste valorisation based on biological routes of specialised microorganisms able to leach solid-containing metals. Because of the physiology of these microorganisms, microbial leaching can be economically feasible, besides being an environmentally sustainable process. Like Bacteria and Fungi, Archaea are also capable of metal leaching activity, though their potential is underestimated. Among them, the extremophiles are the most studied and applied in the field of metal recovery, while mesophilic species are less common but still of high interest. Here we provide the state of industrial application of bio-metallurgy and report on the state of the art of Archaea exploitation in metal recovery from e-waste. Moreover, we give a special highlight to methanogenic archaea, which are able to convert CO2 into methane in order to highlight the potential for the valorisation of CO2-rich industrial streams generated by key processes (i.e., anaerobic digestion, concrete, and steel production) in CH4 for gas grid distribution, while making metals content in e-waste available again as raw material

The physiology of trace elements in biological methane production

Trace element (TE) requirements of Methanothermobacter okinawensis and Methanothermobacter marburgensis were examined in silico, and using closed batch and fed-batch cultivation experiments. In silico analysis revealed genomic differences among the transport systems and enzymes related to the archaeal Wood-Ljungdahl pathway of these two methanogens. M. okinawensis responded to rising concentrations of TE by increasing specific growth rate (µ) and volumetric productivity (MER) during closed batch cultivation, and can grow and produce methane (CH4) during fed-batch cultivation. M. marburgensis showed higher µ and MER during fed-batch cultivation and was therefore prioritized for subsequent optimization of CO2-based biological CH4 production. Multiple-parameter cultivation dependency on growth and productivity of M. marburgensis was finally examined using exponential fed-batch cultivation at different medium-, TE- and sulphide dilution rates, and different gas inflow rates. MER of 476 mmol L−1 h−1 and µ of 0.69 h−1 were eventually obtained during exponential fed-batch cultivations employing M. marburgensis