On the pathways feeding the H₂ production process in nutrient-replete, hypoxic conditions : commentary on the article 'Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures', by Jurado-Oller et al., Biotechnology for Biofuels, published September 7, 2015; 8:149

Background: Under low O-2 concentration ( hypoxia) and low light, Chlamydomonas cells can produce H-2 gas in nutrient-replete conditions. This process is hindered by the presence of O-2, which inactivates the [FeFe]-hydrogenase enzyme responsible for H-2 gas production shifting algal cultures back to normal growth. The main pathways accounting for H-2 production in hypoxia are not entirely understood, as much as culture conditions setting the optimal redox state in the chloroplast supporting long-lasting H-2 production. The reducing power for H-2 production can be provided by photosystem II (PSII) and photofermentative processes during which proteins are degraded via yet unknown pathways. In hetero- or mixotrophic conditions, acetate respiration was proposed to indirectly contribute to H-2 evolution, although this pathway has not been described in detail. Main body: Recently, Jurado-Oller et al. (Biotechnol Biofuels 8: 149, 7) proposed that acetate respiration may substantially support H-2 production in nutrient-replete hypoxic conditions. Addition of low amounts of O-2 enhanced acetate respiration rate, particularly in the light, resulting in improved H-2 production. The authors surmised that acetate oxidation through the glyoxylate pathway generates intermediates such as succinate and malate, which would be in turn oxidized in the chloroplast generating FADH(2) and NADH. The latter would enter a PSII-independent pathway at the level of the plastoquinone pool, consistent with the light dependence of H-2 production. The authors concluded that the water-splitting activity of PSII has a minor role in H-2 evolution in nutrient-replete, mixotrophic cultures under hypoxia. However, their results with the PSII inhibitor DCMU also reveal that O-2 or acetate additions promoted acetate respiration over the usually dominant PSII-dependent pathway. The more oxidized state experienced by these cultures in combination with the relatively short experimental time prevented acclimation to hypoxia, thus precluding the PSII-dependent pathway from contributing to H-2 production. Conclusions: In Chlamydomonas, continuous H-2 gas evolution is expected once low O-2 partial pressure and optimal reducing conditions are set. Under nutrient-replete conditions, the electrogenic processes involved in H-2 photoproduction may rely on various electron transport pathways. Understanding how physiological conditions select for specific metabolic routes is key to achieve economic viability of this renewable energy source

Osmotic stress confers enhanced cell integrity to hydrostatic pressure but impairs growth in Alcanivorax borkumensis SK2

Alcanivorax is a hydrocarbonoclastic genus dominating oil spills worldwide. While its presence has been detected in oil-polluted seawaters, marine sediment and salt marshes under ambient pressure, its presence in deep-sea oil-contaminated environments is negligible. Recent laboratory studies highlighted the piezosensitive nature of some Alcanivorax species, whose growth yields are highly impacted by mild hydrostatic pressures (HPs). In the present study, osmotic stress was used as a tool to increase HP resistance in the type strain Alcanivorax borkumensis SK2. Control cultures grown under standard conditions of salinity and osmotic pressure with respect to seawater (35.6 ppt or 1136 mOsm kg(-1), respectively) were compared with cultures subjected to hypo- and hyperosmosis (330 and 1720 mOsm kg(-1), or 18 and 62 ppt in salinity, equivalent to brackish and brine waters, respectively), under atmospheric or increased HP (0.1 and 10 MPa). Osmotic stress had a remarkably positive impact on cell metabolic activity in terms of CO2 production (thus, oil bioremediation) and O-2 respiration under hyperosmosis, as acclimation to high salinity enhanced cell activity under 10 MPa by a factor of 10. Both osmotic shocks significantly enhanced cell protection by reducing membrane damage under HP, with cell integrities close to 100% under hyposmosis. The latter was likely due to intracellular water-reclamation as no trace of the piezolyte ectoine was found, contrary to hyperosmosis. Notably, ectoine production was equivalent at 0.1 MPa in hyperosmosis-acclimated cells and at 10 MPa under isosmotic conditions. While stimulating cell metabolism and enhancing cell integrity, osmotic stress had always a negative impact on culture growth and performance. No net growth was observed during 4-days incubation tests, and CO2:O-2 ratios and pH values indicated that culture performance in terms of hydrocarbon degradation was lowered by the effects of osmotic stress alone or combined with increased HP. These findings confirm the piezosensitive nature of A. borkumensis, which lacks proper resistance mechanisms to improve its metabolic efficiency under increased HP, thus explaining its limited role in oil-polluted deep-sea environments

Efficient molasses fermentation under high salinity by inocula of marine and terrestrial origin

BACKGROUND: Molasses is a dense and saline by-product of the sugar agroindustry. Its high organic content potentially fuels a myriad of renewable products of industrial interest. However, the biotechnological exploitation of molasses is mainly hampered by the high concentration of salts, an issue that is nowadays tackled through dilution. In the present study, the performance of microbial communities derived from marine sediment was compared to that of communities from a terrestrial environment (anaerobic digester sludge). The aim was to test whether adaptation to salinity represented an advantage for fermenting molasses into renewable chemicals such as volatile fatty acids (VFAs) although high sugar concentrations are uncommon to marine sediment, contrary to anaerobic digesters. RESULTS: Terrestrial and marine microbial communities were enriched in consecutive batches at different initial pH values (pH(i); either 6 or 7) and molasses dilutions (equivalent to organic loading rates (OLRs) of 1 or 5 g(COD) L(−1) d(−1)) to determine the best VFA production conditions. Marine communities were supplied with NaCl to maintain their native salinity. Due to molasses inherent salinity, terrestrial communities experienced conditions comparable to brackish or saline waters (20–47 mS cm(−1)), while marine conditions resembled brine waters (>47 mS cm(−1)). Enrichments at optimal conditions of OLR 5 g(COD) L(-1) d(-1) and pH(i) 7 were transferred into packed-bed biofilm reactors operated continuously. The reactors were first operated at 5 g(COD) L(-1) d(-1), which was later increased to OLR 10 g(COD) L(−1) d(−1). Terrestrial and marine reactors had different gas production and community structures but identical, remarkably high VFA bioconversion yields (above 85%) which were obtained with conductivities up to 90 mS cm(−1). COD-to-VFA conversion rates were comparable to the highest reported in literature while processing other organic leftovers at much lower salinities. CONCLUSIONS: Although salinity represents a major driver for microbial community structure, proper acclimation yielded highly efficient systems treating molasses, irrespective of the inoculum origin. Selection of equivalent pathways in communities derived from different environments suggests that culture conditions select for specific functionalities rather than microbial representatives. Mass balances, microbial community composition, and biochemical analysis indicate that biomass turnover rather than methanogenesis represents the main limitation to further increasing VFA production with molasses. This information is relevant to moving towards molasses fermentation to industrial application. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-017-0701-8) contains supplementary material, which is available to authorized users

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 beta-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

Reduced TCA cycle rates at high hydrostatic pressure hinder hydrocarbon degradation and obligate oil degraders in natural, deep-sea microbial communities

Petroleum hydrocarbons reach the deep-sea following natural and anthropogenic factors. The process by which they enter deep-sea microbial food webs and impact the biogeochemical cycling of carbon and other elements is unclear. Hydrostatic pressure (HP) is a distinctive parameter of the deep sea, although rarely investigated. Whether HP alone affects the assembly and activity of oil-degrading communities remains to be resolved. Here we have demonstrated that hydrocarbon degradation in deep-sea microbial communities is lower at native HP (10 MPa, about 1000 m below sea surface level) than at ambient pressure. In long-term enrichments, increased HP selectively inhibited obligate hydrocarbon-degraders and downregulated the expression of beta-oxidation-related proteins (i.e., the main hydrocarbon-degradation pathway) resulting in low cell growth and CO2 production. Short-term experiments with HP-adapted synthetic communities confirmed this data, revealing a HP-dependent accumulation of citrate and dihydroxyacetone. Citrate accumulation suggests rates of aerobic oxidation of fatty acids in the TCA cycle were reduced. Dihydroxyacetone is connected to citrate through glycerol metabolism and glycolysis, both upregulated with increased HP. High degradation rates by obligate hydrocarbon-degraders may thus be unfavourable at increased HP, explaining their selective suppression. Through lab-scale cultivation, the present study is the first to highlight a link between impaired cell metabolism and microbial community assembly in hydrocarbon degradation at high HP. Overall, this data indicate that hydrocarbons fate differs substantially in surface waters as compared to deep-sea environments, with in situ low temperature and limited nutrients availability expected to further prolong hydrocarbons persistence at deep sea

Physiology and Biotechnology of the Hydrogen Production with the Green Microalga Chlamydomonas reinhardtii

The hydrogen production in the green microalga Chlamydomonas reinhardtii was evaluated by means of a detailed physiological and biotechnological study. First, a wide screening of the hydrogen productivity was done on 22 strains of C. reinhardtii, most of which mutated at the level of the D1 protein. The screening revealed for the first time that mutations upon the D1 protein may result on an increased hydrogen production. Indeed, productions ranged between 0 and more than 500 mL hydrogen per liter of culture (Torzillo, Scoma et al., 2007a), the highest producer (L159I-N230Y) being up to 5 times more performant than the strain cc124 widely adopted in literature (Torzillo, Scoma, et al., 2007b). Improved productivities by D1 protein mutants were generally a result of high photosynthetic capabilities counteracted by high respiration rates. Optimization of culture conditions were addressed according to the results of the physiological study of selected strains. In a first step, the photobioreactor (PBR) was provided with a multiple-impeller stirring system designed, developed and tested by us, using the strain cc124. It was found that the impeller system was effectively able to induce regular and turbulent mixing, which led to improved photosynthetic yields by means of light/dark cycles. Moreover, improved mixing regime sustained higher respiration rates, compared to what obtained with the commonly used stir bar mixing system. As far as the results of the initial screening phase are considered, both these factors are relevant to the hydrogen production. Indeed, very high energy conversion efficiencies (light to hydrogen) were obtained with the impeller device, prooving that our PBR was a good tool to both improve and study photosynthetic processes (Giannelli, Scoma et al., 2009). In the second part of the optimization, an accurate analysis of all the positive features of the high performance strain L159I-N230Y pointed out, respect to the WT, it has: (1) a larger chlorophyll optical cross-section; (2) a higher electron transfer rate by PSII; (3) a higher respiration rate; (4) a higher efficiency of utilization of the hydrogenase; (5) a higher starch synthesis capability; (6) a higher per cell D1 protein amount; (7) a higher zeaxanthin synthesis capability (Torzillo, Scoma et al., 2009). These information were gathered with those obtained with the impeller mixing device to find out the best culture conditions to optimize productivity with strain L159I-N230Y. The main aim was to sustain as long as possible the direct PSII contribution, which leads to hydrogen production without net CO2 release. Finally, an outstanding maximum rate of 11.1 ± 1.0 mL/L/h was reached and maintained for 21.8 ± 7.7 hours, when the effective photochemical efficiency of PSII (ΔF/F'm) underwent a last drop to zero. If expressed in terms of chl (24.0 ± 2.2 µmoles/mg chl/h), these rates of production are 4 times higher than what reported in literature to date (Scoma et al., 2010a submitted). DCMU addition experiments confirmed the key role played by PSII in sustaining such rates. On the other hand, experiments carried out in similar conditions with the control strain cc124 showed an improved final productivity, but no constant PSII direct contribution. These results showed that, aside from fermentation processes, if proper conditions are supplied to selected strains, hydrogen production can be substantially enhanced by means of biophotolysis. A last study on the physiology of the process was carried out with the mutant IL. Although able to express and very efficiently utilize the hydrogenase enzyme, this strain was unable to produce hydrogen when sulfur deprived. However, in a specific set of experiments this goal was finally reached, pointing out that other than (1) a state 1-2 transition of the photosynthetic apparatus, (2) starch storage and (3) anaerobiosis establishment, a timely transition to the hydrogen production is also needed in sulfur deprivation to induce the process before energy reserves are driven towards other processes necessary for the survival of the cell. This information turned out to be crucial when moving outdoor for the hydrogen production in a tubular horizontal 50-liter PBR under sunlight radiation. First attempts with laboratory grown cultures showed that no hydrogen production under sulfur starvation can be induced if a previous adaptation of the culture is not pursued outdoor. Indeed, in these conditions the hydrogen production under direct sunlight radiation with C. reinhardtii was finally achieved for the first time in literature (Scoma et al., 2010b submitted). Experiments were also made to optimize productivity in outdoor conditions, with respect to the light dilution within the culture layers. Finally, a brief study of the anaerobic metabolism of C. reinhardtii during hydrogen oxidation has been carried out. This study represents a good integration to the understanding of the complex interplay of pathways that operate concomitantly in this microalga

Effect of hydraulic retention time on biohydrogen and volatile fatty acids production during acidogenic digestion of dephenolized olive mill wastewaters

The influence of Hydraulic Retention Time (HRT) on the performances of a recently developed biotechnological anaerobic acidogenic process fed with dephenolized Olive Mill Wastewater (OMW) was investigated. The study was carried out under mesophilic conditions in Packed Bed Biofilm Reactors (PBBRs), filled with ceramic cubes and inoculated with a characterized and acclimated acidogenic microbial consortium. The PBBRs were fed with a HRT of 7, 5, 3 or 1 day, which corresponded to Organic Loading Rates (OLRs) of about 5.5, 7.8, 12.9 and 38.8 g L-1 d-1, respectively. A significant production of a H2-rich biogas was observed when shorter HRTs were applied: in particular, H2 relative amount and productivity increased from 3% to 32% and from 0.20 to 6.10 dm3 m3 h1, respectively, by decreasing the HRT from 7 to 1 day. On the contrary, shorter HRTs turned into a lower accumulation of Volatile Fatty Acids (VFAs), whose highest amounts were found with HRTs of 7 and 5 days (about 18.4 and 19.7 g L-1 COD equivalents, respectively). The highest conversion yield of COD into VFAs (36%) was obtained with a HRT of 5 days, when VFAs represented about 78% of the effluent COD. HRT also influenced the composition of the VFA mixture: acetic, propionic and butyric acid were the most prominent VFAs, being their relative amounts higher when PBBRs were operated with shorter HRTs (up to 19, 12 and 42% of the whole mixture, respectively, when HRT was 1 day)

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 mu mol photons m(-2) s(-1) in combination(-1) with chlorophyll concentrations of 12 and 24 mg(-2) L-1. A high light intensity (140 mu mol photons in 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 mu mol 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