Alberto Scoma

Sustained H2 production in a Chlamydomonas reinhardtii D1 protein mutant

In the present investigation, a detailed biochemical analysis of the high H₂ producer D1 protein mutant strain L159I-N230Y of Chlamydomonas reinhardtii, carrying a double amino acid substitution, was made. The leucine residue L159 was replaced by isoleucine, and the N230 asparagine was replaced by tyrosine. The performance of this strain was compared to that of the cc124 strain. The mutant showed a sustained capacity to donate electrons by means of direct biophotolysis for H₂ production, as demonstrated by the higher efficiency of utilization of the hydrogenase enzyme when carried out under anaerobic conditions. The latter property was maintained also under sulfur deprivation. Furthermore, when compared to the cc124, the mutant showed a higher amount of D1 protein content, a higher carbohydrate storage capacity and a sustained PSII direct contribution to the H₂ production during sulfur deprivation. The addition of DCMU to the cells showed that as much as 7.0 mL H₂ liter of culture h⁻¹ were produced by means of direct biophotolysis. The maximum apparent light-to-hydrogen conversion efficiency expressed on PAR (photosynthetically active radiation) reached 3.22%, while PSII efficiency to perform direct biophotolysis was calculated to be 2.03%. These values are significantly higher than what has been reported in the literature.

Interplay between light intensity, chlorophyll concentration and culture mixing on the hydrogen production in sulfur‐deprived Chlamydomonas reinhardtii cultures grown in laboratory photobioreactors

Relationships between light intensity and chlorophyll concentration on hydrogen production were investigated in a sulfur‐deprived Chlamydomonas reinhardtii culture in a laboratory scale photobioreactor (PBR) equipped with two different stirring devices. In the first case, the culture was mixed using a conventional magnetic stir bar, while in the second it was mixed using an impeller equipped with five turbines. Experiments were carried out at 70 and 140 µmol photons m−2 s−1 in combination with chlorophyll concentrations of 12 and 24 mg L−1. A high light intensity (140 µmol photons m−2 s−1, supplied on both sides of the PBR) in combination with a low chlorophyll concentration (12 mg L−1) inhibited the production of hydrogen, in particular in the culture mixed with the stir bar. An optimal combination for hydrogen production was found when the cultures were exposed to 140 µmol photons m−2 s−1 (on both sides) and 24 mg L−1 of chlorophyll. Under these conditions, the hydrogen production output rate reached about 120 mL L−1 in the culture mixed with the stir bar, and rose to about 170 mL L−1 in the one mixed with the impeller. These outputs corresponded to a mean light conversion efficiency of 0.56% and 0.81%, respectively. However, the efficiency increased to 1.08% and 1.64%, respectively, when maximum hydrogen rates were considered. The better performance of the dense cultures mixed with an impeller was mainly attributed to an intermittent illumination pattern to which the cells were subjected (time cycles within 50–100 ms) which influenced the hydrogen production (1) directly, by providing the PSII with a higher production of electrons for the hydrogenase and (2) indirectly, through a higher synthesis of carbohydrates. The fluid dynamics in the PBR equipped with the impeller was characterized. The better mixing state achieved in the PBR of the new configuration makes it a useful tool for studying the hydrogen production process involving photosynthetic microorganisms, and provides a better insight into the physiology of the process. Biotechnol. Bioeng. 2009; 104: 76–90

Anaerobic acidogenic digestion of olive mill wastewaters in biofilm reactors packed with ceramic filters or granular activated carbon

Four identically configured anaerobic packed bed biofilm reactors were developed and employed in the continuous acidogenic digestion of olive mill wastewaters to produce volatile fatty acids (VFAs), which can be exploited in the biotechnological production of polyhydroxyalkanoates. Ceramic porous cubes or granular activated carbon were used as biofilm supports. Aside packing material, the role of temperature and organic loading rate (OLR) on VFA production yield and mixture composition were also studied. The process was monitored through a chemical, microbiological and molecular biology integrated procedure. The highest wastewater acidification yield was achieved with the ceramic-based technology at 25 degrees C, with an inlet COD and an OLR of about 17 g/L and 13 g/L/day, respectively. Under these conditions, about the 66% of the influent COD (not including its VFA content) was converted into VFAs, whose final amount represented more than 82% of the influent COD. In particular, acetic, propionic and butyric acids were the main VFAs by composing the 55.7, 21.5 and 14.4%, respectively, of the whole VFA mixture. Importantly, the relative concentrations of acetate and propionate were affected by the OLR parameter. The nature of the packing material remarkable influenced the process performances, by greatly affecting the biofilm bacterial community structure. In particular, ceramic cubes favoured the immobilization of Firmicutes of the genera Bacillus, Paenibacillus and Clostridium, which were probably involved in the VFA producing process.

A physicochemical–biotechnological approach for an integrated valorization of olive mill wastewater

An integrated physicochemical-biotechnological approach for a multipurpose valorization of olive mill wastewaters was studied. More than 60% of the wastewater natural polyphenols were recovered through a solid phase extraction procedure, by employing Amberlite XAD16 resin as the adsorbent and ethanol as the biocompatible desorbing phase. Thereafter, the dephenolized effluent was fed to a mesophilic anaerobic acidogenic packed-bed biofilm reactor for the bioconversion of the organic leftover into volatile fatty acids (VFAs). A VFAs concentration of 19 gCODL(-1) was obtained, representing more than 70% of the COD occurring in the anaerobic effluent. The biotechnological process was assessed by means of bio-molecular analyses, which showed that the reactor packed bed was mostly colonized by bacteria of the Firmicutes phylogenetic group. The biorefinery scheme developed in this study allowed the obtainment of 1.59 g of polyphenols per liter of wastewater treated and 2.72 gCODL(-1) day(-1) of VFAs.

Outdoor H2 production in a 50-L tubular photobioreactor by means of a sulfur-deprived culture of the microalga Chlamydomonas reinhardtii

In the past decade, H₂ production using the green microalga Chlamydomonas reinhardtii has been extensively studied under laboratory-scale photobioreactors, while information on outdoor cultures is still lacking. In this paper, the results of experiments conducted with sulfur-deprived cultures of C. reinhardtii carried out in a 50-L horizontal tubular photobioreactor are presented. Hydrogen production experiments were carried out under both artificial and direct solar light. In both cases, the H₂ output attained was 18-20% of what obtained in the laboratory. However, no significant changes in the H₂ production were observed when cells grown outdoors were tested under laboratory conditions. Chlorophyll fluorescence measurements showed that outdoor cultures were subjected to strong photo-inhibition, due to the combination of high solar light intensity and sulfur-deprivation. Indeed, H₂ production was only achieved outdoors when cultures were previously acclimated to sunlight, a condition that caused a number of physiological changes, namely: (i) a decrease in the chlorophyll content per unit of dry weight; (ii) an increase in the photosynthesis and respiration rates, and (iii) a higher induction of the xanthophyll cycle pigments as compared to non-acclimated cultures. It was concluded that the reduced H₂ output achieved in the 50-L photobioreactor was due to the different illumination pattern to which the cultures were exposed (one-sided vs. two-sided illumination provided in the laboratory), as well as to the great difference in the mixing times (60 min vs. 15.5s achieved in the lab-scale photobioreactor). To the very best of our knowledge this is the first time that H₂ production with green algae has been achieved by means of solar light.

Microbial oil-degradation under mild hydrostatic pressure (10 MPa): which pathways are impacted in piezosensitive hydrocarbonoclastic bacteria?

Oil spills represent an overwhelming carbon input to the marine environment that immediately impacts the sea surface ecosystem. Microbial communities degrading the oil fraction that eventually sinks to the seafloor must also deal with hydrostatic pressure, which linearly increases with depth. Piezosensitive hydrocarbonoclastic bacteria are ideal candidates to elucidate impaired pathways following oil spills at low depth. In the present paper, we tested two strains of the ubiquitous Alcanivorax genus, namely A. jadensis KS_339 and A. dieselolei KS_293, which is known to rapidly grow after oil spills. Strains were subjected to atmospheric and mild pressure (0.1, 5 and 10 MPa, corresponding to a depth of 0, 500 and 1000 m, respectively) providing n-dodecane as sole carbon source. Pressures equal to 5 and 10 MPa significantly lowered growth yields of both strains. However, in strain KS_293 grown at 10 MPa CO2 production per cell was not affected, cell integrity was preserved and PO43− uptake increased. Analysis of its transcriptome revealed that 95% of its genes were downregulated. Increased transcription involved protein synthesis, energy generation and respiration pathways. Interplay between these factors may play a key role in shaping the structure of microbial communities developed after oil spills at low depth and limit their bioremediation potential.

High impact biowastes from South European agro-industries as feedstock for second-generation biorefineries

Abstract Availability of bio-based chemicals, materials and energy at reasonable cost will be one of the forthcoming issues for the EU economy. In particular, the development of technologies making use of alternative resources to fossil fuels is encouraged by the current European research and innovation strategy to face the societal challenge of natural resource scarcity, fossil resource dependence and sustainable economic growth. In this respect, second- generation biorefineries, i.e. biorefineries fed with biowastes, appear to be good candidates to substitute and replace the present downstream processing scheme. Contrary to first-generation biorefineries, which make use of dedicated crops or primary cultivations to achieve such a goal, the former employ agricultural, industrial, zootechnical, fishery and forestry biowastes as the main feedstock. This leaves aside any ethical and social issue generated by first-generation approaches, and concomitantly prevents environmental and economical issues associated with the disposal of the aforementioned leftovers. Unfortunately, to date, a comprehensive and updated mapping of the availability and potential use of bioresources for second-generation biorefineries in Europe is missing. This is a lack that severely limits R&D and industrial applications in the sector. On the other hand, attempts at valorizing the most diverse biowastes dates back to the nineteenth century and plenty of information in the literature on their sustainable exploitation is available. However, the large majority of these investigations have been focused on single fractions of biowastes or single steps of biowaste processing, preventing considerations on an integrated and modular (cascade) approach for the whole valorization of organic leftovers. This review aims at addressing these issues by gathering recent data on (a) some of the main high-impact biowastes located in Europe and in particular in its Southern part, and (b) the bio-based chemicals, materials and fuels that can be produced from such residues. In particular, we focused on those key compounds referred to as “chemical platforms”, which have been indicated as fundamental to generate the large majority of the industrially relevant goods to date.

Challenging Oil Bioremediation at Deep-Sea Hydrostatic Pressure.

The Deepwater Horizon accident has brought oil contamination of deep-sea environments to worldwide attention. The risk for new deep-sea spills is not expected to decrease in the future, as political pressure mounts to access deep-water fossil reserves, and poorly tested technologies are used to access oil. This also applies to the response to oil-contamination events, with bioremediation the only (bio)technology presently available to combat deep-sea spills. Many questions about the fate of petroleum-hydrocarbons within deep-sea environments remain unanswered, as well as the main constraints limiting bioremediation under increased hydrostatic pressures and low temperatures. The microbial pathways fueling oil bioassimilation are unclear, and the mild upregulation observed for beta-oxidation-related genes in both water and sediments contrasts with the high amount of alkanes present in the spilled oil. The fate of solid alkanes (tar), hydrocarbon degradation rates and the reason why the most predominant hydrocarbonoclastic genera were not enriched at deep-sea despite being present at hydrocarbon seeps at the Gulf of Mexico have been largely overlooked. This mini-review aims at highlighting the missing information in the field, proposing a holistic approach where in situ and ex situ studies are integrated to reveal the principal mechanisms accounting for deep-sea oil bioremediation.

An impaired metabolic response to hydrostatic pressure explains Alcanivorax borkumensis recorded distribution in the deep marine water column.

Alcanivorax borkumensis is an ubiquitous model organism for hydrocarbonoclastic bacteria, which dominates polluted surface waters. Its negligible presence in oil-contaminated deep waters (as observed during the Deepwater Horizon accident) raises the hypothesis that it may lack adaptive mechanisms to hydrostatic pressure (HP). The type strain SK2 was tested under 0.1, 5 and 10 MPa (corresponding to surface water, 500 and 1000 m depth, respectively). While 5 MPa essentially inactivated SK2, further increase to 10 MPa triggered some resistance mechanism, as indicated by higher total and intact cell numbers. Under 10 MPa, SK2 upregulated the synthetic pathway of the osmolyte ectoine, whose concentration increased from 0.45 to 4.71 fmoles cell−1. Central biosynthetic pathways such as cell replication, glyoxylate and Krebs cycles, amino acids metabolism and fatty acids biosynthesis, but not β-oxidation, were upregulated or unaffected at 10 MPa, although total cell number was remarkably lower with respect to 0.1 MPa. Concomitantly, expression of more than 50% of SK2 genes was downregulated, including genes related to ATP generation, respiration and protein translation. Thus, A. borkumensis lacks proper adaptation to HP but activates resistance mechanisms. These consist in poorly efficient biosynthetic rather than energy-yielding degradation-related pathways, and suggest that HP does represent a major driver for its distribution at deep-sea.

Biotechnologies for Marine Oil Spill Cleanup: Indissoluble Ties with Microorganisms

The ubiquitous exploitation of petroleum hydrocarbons (HCs) has been accompanied by accidental spills and chronic pollution in marine ecosystems, including the deep ocean. Physicochemical technologies are available for oil spill cleanup, but HCs must ultimately be mineralized by microorganisms. How environmental factors drive the assembly and activity of HC-degrading microbial communities remains unknown, limiting our capacity to integrate microorganism-based cleanup strategies with current physicochemical remediation technologies. In this review, we summarize recent findings about microbial physiology, metabolism and ecology and describe how microbes can be exploited to create improved biotechnological solutions to clean up marine surface and deep waters, sediments and beaches.