This paper reports the first characterization of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron-sulfur centres involved in electron transfer and the catalytic center H-cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H2 evolution at high rate of 1645 ± 16 s(-1) . The optimal conditions for catalysis are in the pH range 6.5-8.0 and at the temperature of 50 °C. EPR spectroscopy showed that the H-cluster of the oxidized enzyme displays a spectrum coherent with the Hox state, whereas the CO-inhibited enzyme has a spectrum coherent with the Hox -CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox ; upon reduction with H2 , vibrational modes assigned to the Hred state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the Hox -CO state. The spectroscopic features observed are similar to those of the [FeFe]-hydrogenases class, but relevant differences were observed given the different protein environment hosting the H-cluster.

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 environmental-like 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.

SummaryHuman activity has been altering many ecological cycles for decades, disturbing the natural mechanisms which are responsible for re‐establishing the normal environmental balances. Probably, the most disrupted of these cycles is the cycle of carbon. In this context, many technologies have been developed for an efficient CO 2 removal from the atmosphere. Once captured, it could be stored in large geological formations and other reservoirs like oceans. This strategy could present some environmental and economic problems. Alternately, CO 2 can be transformed into carbonates or different added‐value products, such as biofuels and bioplastics, recycling CO 2 from fossil fuel. Currently different methods are being studied in this field. We classified them into biological, inorganic and hybrid systems for CO 2 transformation. To be environmentally compatible, they should be powered by renewable energy sources. Although hybrid systems are still incipient technologies, they have made great advances in the recent years. In this scenario, biotechnology is the spearhead of ambitious strategies to capture CO 2 and reduce global warming.

Background: Poly(3-hydroxybutyrate) (PHB) is a polymer produced by Azotobacter vinelandii. The production of PHB in a bioreactor is affected by oxygen transfer conditions. The aim of this study was to evaluate PHB synthesis in extended batch of A. vinelandii, to obtain an operation curve (productivity versus oxygen transfer rate) and to scale up the bioprocess. Results: PHB production by A. vinelandii under dinitrogen fixation was evaluated using an extended batch modality under different oxygen transfer rates (OTRs). Extended batch cultures were performed using different agitation rates (400, 600, 800 and 1000 rpm), which determined different OTRs in the cultures. Under the conditions evaluated, it was possible to establish an operation curve of PHB productivity at different OTRs, allowing for the establishment of the maximum productivity of PHB. The maximum PHB productivity obtained was 0.40 ± 0.05 g L −1 h −1 , which was reached under an OTR of 22.1 ± 1.5 mmol L −1 h −1. Using the OTR as a criterion to scale up PHB production from 3 to 30 L, it was possible to obtain a similar PHB concentration (7.9 ± 0.0 g L −1) and volumetric productivity (0.43 ± 0.04 g L −1 h −1) on a 30 L scale. Conclusions: The results lead to the conclusion that in extended cultures and under dinitrogen fixation in A. vinelandii cultures, the OTR value determines the maximum PHB productivity. This study provides evidence that the OTR is an adequate parameter as a criterion for the successful scaling up of PHB synthesis for the first time.

Rhodospirillum rubrum is a gram-negative bacterium that naturally takes advantage of CO and which, in the presence of acetate, accumulates carbon and energy units as polyhydroxybutyrate (PHB). Since the conversion of CO depends on a large protein membrane complex that is expressed after the exposure to carbon monoxide, this study presents the effects of a CO-based acclimation in R. rubrum on the growth trend and on the production of PHB. The strain was cultured in two consecutive fermentation cycles on 15% of CO, and the behaviour of this species, in the presence of acetate or a reducing sugar, such as fructose, was compared. The exposure of R. rubrum to CO during the first adaptation phase led to the development of a metabolically active population characterised by a greater biomass growth. The supply of fructose ensured a shorter lag-phase and a higher biomass titre, but it also determined a decrease in the biopolymer accumulation. However, R. rubrum showed the best carbon utilisation in the absence of fructose, with a growth molar yield of 48 mg mol −1 , compared to the 12 mg mol −1 obtained for fructose feeding.