Fixing N2 into cyanophycin: continuous cultivation of Nostoc sp. PCC 7120.

Two diazotrophic cyanobacteria (Anabaena cylindrica PCC 7122 and Nostoc sp. PCC 7120) were cultivated to produce cyanophycin, a nitrogen reserve compound, under nitrogen fixing conditions. In preliminary continuous experiments, Nostoc sp. was shown to be more efficient, accumulating a higher amount of cyanophycin and showing a greater capability to fix atmospheric nitrogen in the biomass (67 mgN d-1 of fixed nitrogen per liter of culture). The operating conditions were then optimized to maximize the cyanophycin productivity: the effect of incident light intensity, residence time and nitrogen availability were investigated. Nitrogen availability and/or pH played a major role with respect to biomass production, whereas phosphorus limitation was the main variable to maximize cyanophycin accumulation. In this way, it was possible to achieve a stable and continuous production of cyanophycin (CGP) under diazotrophic conditions, obtaining a maximum cyanophycin productivity of 15 mgCGP L-1 d-1. KEY POINTS: • Diazotrophic cyanobacteria produce stable amount of cyanophycin in continuous PBR. • Nostoc sp. proved to be more efficient in producing cyanophycin than Anabaena sp. • P deprivation is the major variable to increase cyanophycin productivity in continuous.

Mixotrophy in Synechocystis sp. for the treatment of wastewater with high nutrient content: effect of CO2 and light.

Synechocystis sp. has remarkable mixotrophic capabilities, as well as an efficient exploitation of nitrogen and phosphorus, that may be applied in wastewater treatment based on cyanobacteria. To better investigate the exploitation of algal mixotrophy in bioremediation, this species was used in axenic respirometric tests to ascertain the effect of high light and non limiting CO2 supply on the overall regulation of mixotrophy, resulting in an inhibition of the exploitation of organic carbon. The same species was then cultured in real, unsterilized effluent obtained from the acidogenic fermentation of sludge, which contains a high concentration of nutrients (approximately 600, 90 and 6000 mg L-1 of N, P and COD, respectively) and it is often inhibiting for many microalgal species. On the contrary, Synechocystis sp., showed a remarkable growth and a removal up to 96% of phosphorus, 66% of nitrogen and of 68% of COD in such a complex waste stream.

Respirometry as a tool to quantify kinetic parameters of microalgal mixotrophic growth.

Modeling microalgal mixotrophy is challenging, as the regulation of algal metabolism is affected by many environmental factors. A reliable tool to simulate microalgal behavior in complex systems, such as wastewaters, may help in setting the proper values of operative variables, provided that model parameters have been properly evaluated. In this work, a new respirometric protocol is proposed to quickly obtain the half-saturation constant values for several nutrients. The protocol was first verified for autotrophic exploitation of ammonium and phosphorus (Monod kinetics), as well as of light intensity (Haldane model), further elaborated on specific light supply basis. It was then applied to measure the kinetic parameters of heterotrophic growth. The half-saturation constants for nitrogen and phosphorus resulted comparable with autotrophic ones. The dependence on acetate and dissolved oxygen concentration was assessed. Mixotrophy was modeled as the combination of autotrophic/heterotrophic reactions, implemented in AQUASIM, and validated on batch curves with/without bubbling, under nutrient limitation, and different light intensities. It was shown that the reliability of the proposed respirometric protocol is useful to measure kinetic parameters for nutrients, and therefore to perform bioprocess simulation.

Exploiting symbiotic interactions between Chlorella protothecoides and Brevundimonas diminuta for an efficient single-step urban wastewater treatment.

The application of microalgal bacteria consortia to the treatment of wastewater is receiving increasing attention, meeting the demand for new green and efficient technologies for water remediation. The specificity of the consortium, however, may strongly affect the performance of the treatment. In fact, even though a general exploitation of the O2/CO2 exchange between microalgae and bacteria is effective, some specific interactions may increase the pollutant removal. With this aim, the co-cultivation of Chlorella protothecoides and Brevundimonas diminuta was tested, with particular attention to the removal capability of nitrogen, phosphorus and chemical oxygen demand (COD) from wastewater. Batch experiments were carried out both for the consortium and, separately, for the bacteria and microalgae alone, in order to compare their performances. B. diminuta showed a remarkable capability for removing organic substances and transforming organic nitrogen to ammonium. C. protothecoides efficiently removed nitrogen and phosphorus. As the specific growth rates of the two organisms are different, the co-cultivation was also carried out also in a continuous system, and the effect of hydraulic retention time (HRT) on the steady-state biomass concentration and nutrient removal efficiency was verified. Residence time was found as the main operating variable for obtaining a significant reduction of pollutants from wastewater.

Light intensity affects the mixotrophic carbon exploitation in Chlorella protothecoides: consequences on microalgae-bacteria based wastewater treatment.

Microalgal-bacteria consortia application on wastewater treatment has been widely studied, but a deeper comprehension of consortium interactions is still lacking. In particular, mixotrophic exploitation of organic compounds by microalgae affects gas (CO2 and O2) exchange between microalgae and bacteria, but it is not clear how environmental conditions may regulate algal metabolism. Using a respirometric-based protocol, we evaluated the combined effect of organic carbon and light intensity on oxygen production and consumption by C. protothecoides, and found that the chemical oxygen demand (COD) was not consumed when incident light increased. Batch experiments under different incident lights, with C. protothecoides alone and in consortium with activated sludge bacteria, confirmed the results obtained by respirometry. Continuous system experiments testing the combined effects of light intensity and residence time confirmed that, under limiting light, mixotrophy is preferred by C. protothecoides, and the nutrient (COD, N, P) removal capability of the consortium is enhanced.

Effect of specific light supply rate on photosynthetic efficiency of Nannochloropsis salina in a continuous flat plate photobioreactor.

In this work, Nannochloropsis salina was cultivated in a continuous-flow flat-plate photobioreactor, working at different residence times and irradiations to study the effect of the specific light supply rate on biomass productivity and photosynthetic efficiency. Changes in residence times lead to different steady-state cell concentrations and specific growth rates. We observed that cultures at steady concentration were exposed to different values of light intensity per cell. This specific light supply rate was shown to affect the photosynthetic status of the cells, monitored by fluorescence measurements. High specific light supply rate can lead to saturation and photoinhibition phenomena if the biomass concentration is not optimized for the selected operating conditions. Energy balances were applied to quantify the biomass growth yield and maintenance requirements in N. salina cells.

Maximizing the production of Scenedesmus obliquus in photobioreactors under different irradiation regimes: experiments and modeling.

Maximizing biomass productivity and photosynthetic efficiency are key factors to develop large-scale microalgae cultivation for biodiesel production. If the photobioreactor (PBR) is not operated under proper conditions, productivity and efficiency values drop considerably. In this work, the growth of Scenedesmus obliquus in continuous flat-panel PBR is considered. Experimental data and simulations were used with the aim of determining suitable working conditions to achieve maximum productivity. Microalgae concentration and productivity have been measured in a continuous 250 mL flat-panel PBR as a function of the space-time τ. Simulations were performed at both low and high irradiance values, with different light regimes (constant light and day-night profiles). Model parameters were optimized based on laboratory-scale experimental data, and the importance of the maintenance energy requirement as a function of light intensity was outlined. The effect of different extent of axial mixing on PBR performances was investigated. Results obtained show how to determine optimum working conditions and how they could be used in the design of a large-scale PBR to achieve maximum microalgal productivity.

Continuous microalgal cultivation in a laboratory-scale photobioreactor under seasonal day-night irradiation: experiments and simulation.

In this work, the production of Scenedesmus obliquus in a continuous flat-plate laboratory-scale photobioreactor (PBR) under alternated day-night cycles was tested both experimentally and theoretically. Variation of light intensity according to the four seasons of the year were simulated experimentally by a tunable LED lamp, and effects on microalgal growth and productivity were measured to evaluate the conversion efficiency of light energy into biomass during the different seasons. These results were used to validate a mathematical model for algae growth that can be applied to simulate a large-scale production unit, carried out in a flat-plate PBR of similar geometry. The cellular concentration in the PBR was calculated in both steady-state and transient conditions, and the value of the maintenance kinetic term was correlated to experimental profiles. The relevance of this parameter was finally outlined.

Design and experimental validation of an optimized microalgae-bacteria consortium for the bioremediation of glyphosate in continuous photobioreactors.

Glyphosate will be banned from Europe by the end of 2022, but its widespread use in the last decades and its persistence in the environment require the development of novel remediation processes. In this work, a bacterial consortium was designed de novo with the aim to remove glyphosate from polluted water, supported by the oxygen produced by a microalgal species. To this goal, bioinformatics tools were employed to identify the bacterial strains from contaminated sources (Pseudomonas stutzeri; Comamonas odontotermitis; Sinomonas atrocyanea) able to express enzymes for glyphosate degradation, while the microalga Chlorella protothecoides was chosen for its known performances in wastewater treatment. To follow a bioaugmentation approach, the designed consortium was cultivated in continuous photobioreactors at increasing glyphosate concentrations, from 5 to 50 mg L-1, to boost its acclimation to the presence of the herbicide and its capacity to remove it from water. C. protothecoides tolerance to glyphosate was verified through batch experiments. Remarkably, steady state conditions were reached and the consortium was able to live as a community in the reactor. The consortium activity was validated in both synthetic and real wastewater, where glyphosate concentration was reduced by about 53% and 79%, respectively, without the detection of aminomethylphosphonic acid formation.

Microalgae growth in ultra-thin steady-state continuous photobioreactors: assessing self-shading effects.

To disclose the net effect of light on microalgal growth in photobioreactors, self-shading and mixing-induced light-dark cycles must be minimized and discerned from the transient phenomena of acclimation. In this work, we performed experiments of continuous microalgal cultivation in small-scale photobioreactors with different thicknesses (from 2 to 35 mm): working at a steady state allowed us to describe the effect of light after acclimation, while the geometry of the reactor was adjusted to find the threshold light path that can discriminate different phenomena. Experiments showed an increased inhibition under smaller culture light paths, suggesting a strong shading effect at thicknesses higher than 8 mm where mixing-induced light-dark cycles may occur. A Haldane-like model was applied and kinetic parameters retrieved, showing possible issues in the scalability of experimental results at different light paths if mixing-induced light-dark cycles are not considered. To further highlight the influence of mixing cycles, we proposed an analogy between small-scale operations with continuous light and PBR operations with pulsed light, with the computation of characteristic parameters from pulsed-light microalgae growth mathematical modeling.

Effect of residence time in continuous photobioreactor on mass and energy balance of microalgal protein production.

There is increasing interest in new protein sources for the food and feed industry and for the agricultural sector, and microalgae are considered a good alternative, having a high protein content and a well-balanced amino acid profile. However, protein production from microalgae presents several unsolved issues, as the biomass composition changes markedly as a function of cultivation operating conditions. Continuous systems, however, may be properly set to boost the accumulation of protein in the biomass, ensuring stable production. Here, two microalgae and two cyanobacterial species were cultivated in continuous operating photobioreactors (PBR) under nonlimiting nutrient conditions, to study the effects of light intensity and residence time on both biomass and protein productivity at steady state. Although light strongly affected biomass growth inside the PBR, the overall protein pool did not vary in response to irradiance. On the other hand, shorter residence times resulted in protein accumulation of up to 68 % in cyanobacteria, in contrast with green algae, where a minor influence of residence time on biomass composition was observed. Energy balance showed that light conversion to protein decreased with light intensity. Protein content was also related to energy costs for cell maintenance. In conclusion, it is shown that residence time is the key variable to increase protein content and yield of protein production, but its effect depends on the specific species.

Bioremediation of Per- and Poly-Fluoroalkyl Substances (PFAS) by Synechocystis sp. PCC 6803: A Chassis for a Synthetic Biology Approach.

One of the main concerns in industrialized countries is represented by per- and poly-fluoroalkyl substances (PFAS), persistent contaminants hardly to be dealt with by conventional wastewater treatment processes. Phyco-remediation was proposed as a green alternative method to treat wastewater. Synechocystis sp. PCC6803 is a unicellular photosynthetic organism candidate for bioremediation approaches based on synthetic biology, as it is able to survive in a wide range of polluted waters. In this work, we assessed the possibility of applying Synechocystis in PFAS-enriched waters, which was never reported in the previous literature. Respirometry was applied to evaluate short-term toxicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), which did not affect growth up to 0.5 and 4 mg L-1, respectively. Continuous and batch systems were used to assess the long-term effects, and no toxicity was highlighted for both compounds at quite high concentration (1 mg L-1). A partial removal was observed for PFOS and PFOA, (88% and 37%, with removal rates of about 0.15 and 0.36 mg L-1 d-1, respectively). Measurements in fractionated biomass suggested a role for Synechocystis in the sequestration of PFAS: PFOS is mainly internalized in the cell, while PFOA is somehow transformed by still unknown pathways. A preliminary bioinformatic search gave hints on transporters and enzymes possibly involved in such sequestration/transformation processes, opening the route to metabolic engineering in the perspective application of this cyanobacterium as a new phyco-remediation tool, based on synthetic biology.

Modeling the oxygen inhibition in microalgae: An experimental approach based on photorespirometry.

Microalgae cultivation has been the object of relevant interest for many industrial applications. Where high purity of the biomass/product is required, closed photobioreactors (PBRs) appear to be the best technological solution. However, as well as cost, the major drawback of closed systems is oxygen accumulation, which is well known to be responsible for growth inhibition. Only a few quantitative approaches have attempted to describe and model oxygen inhibition, which is the result of different biological mechanisms. Here, we have applied a photorespirometric protocol to assess and quantify the effect of high oxygen concentration on photosynthetic production rate. In particular, the effects of light intensity and biomass concentration were assessed, resulting in different maximum inhibitory oxygen concentrations. Literature models available were found not to fully represent experimental data as a function of concentration and light. Accordingly, a new formulation was proposed and validated to describe the photosynthetic rate as a function of external oxygen concentration.

Continuous Cultivation as a Method to Assess the Maximum Specific Growth Rate of Photosynthetic Organisms.

Modeling the growth of photosynthetic organisms is challenging, due to the complex role of light, which can be limiting because of self-shading, or photoinhibiting in the case of high intensities. A case of particular interest is represented by nitrogen-fixing cyanobacteria, whose growth is controlled not only by the light intensity, but also by the availability of atmospheric nitrogen in the liquid medium. The determination of the maximum specific growth rate is often affected by many variables that, in batch growth systems, may change significantly. On the other hand, in a continuous system, once the steady state is reached the values of all the process variables remain constant, including the biomass concentration and the specific light supply rate. In this work, the diazotrophic cyanobacterium Anabaena PCC 7122 was cultivated in continuous photobioreactors, to investigate the role of nitrogen, light and residence time on growth kinetics, and to retrieve the value of the maximum specific growth rate of this organism. In addition, the kinetic parameters for temperature and the half saturation constant for nitrogen (3 mg L-1) were measured by respirometric tests. Based on the results of continuous experiments, the specific maintenance rate was found to depend on the light intensity supplied to the reactor, ranging between 0.5 and 0.8 d-1. All these parameters were used to develop a kinetic model able to describe the biomass growth in autotrophic conditions. The maximum specific growth rate could hence be determined by applying the kinetic model in the material balances of the continuous photobioreactor, and resulted equal to 8.22 ± 0.69 d-1.

Microalgae-bacteria gas exchange in wastewater: how mixotrophy may reduce the oxygen supply for bacteria.

Microalgae-bacteria consortia application to wastewater treatment is considered as a potential and cheap strategy towards a self-sustaining oxygen-carbon dioxide gas exchange. However, microalgae can also carry out mixotrophy, thus reducing the net oxygen production, due to consumption of organic substrates. In this work, respirometric tests were used to quantify the oxygen reduction in the presence of biodegradable COD (chemical oxygen demand), which resulted up to 70%, depending on the biodegradability of the carbon substrate. The implication of mixotrophic metabolism on nutrient removal in urban wastewater was also measured by co-cultivating C. protothecoides with bacteria from activated sludge. To better understand the contribution of different populations, ad hoc experiments under controlled conditions were designed to quantify the nutrient consumption of bacteria and microalgae. Microalgae and bacteria were cultivated together and separately, with and without external bubbling, so to better ascertain the specific role of gas production and nutrient removal. Results showed that microalgae can remove up to 100 and 85% of P and N respectively, but the contribution on COD consumption may affect the net O2 supply to heterotrophic bacteria. However, a mutual COD consumption by microalgae and bacteria was proved by both experimental growth curves and mass balance application, based on stoichiometry experimentally adjusted.

Effects of pH and Carbon Source on Synechococcus PCC 7002 Cultivation: Biomass and Carbohydrate Production with Different Strategies for pH Control.

Synechococcus PCC 7002 is an interesting species in view of industrial production of carbohydrates. The cultivation performances of this species are strongly affected by the pH of the medium, which also influences the carbohydrate accumulation. In this work, different methods of pH control were analyzed, in order to obtain a higher production of both Synechococcus biomass and carbohydrates. To better understand the influence of pH on growth and carbohydrate productivity, manual and automatic pH regulation in CO2 and bicarbonate system were applied. The pH value of 8.5 resulted the best to achieve both of these goals. From an industrial point of view, an alternative way to maintain the pH practically constant during the entire period of cultivation is the exploitation of the bicarbonate-CO2 buffer system, with the double aim to maintain the pH in the viability range and also to provide the amount of carbon required by growth. In this condition, a high concentration of biomass (6 g L-1) and carbohydrate content (around 60 %) were obtained, which are promising in view of a potential use for bioethanol production. The chemical equilibrium of C-N-P species was also evaluated by applying the ionic balance equations, and a relation between the sodium bicarbonate added in the medium and the equilibrium value of pH was discussed.

Cultivation of Scenedesmus obliquus in liquid hydrolysate from flash hydrolysis for nutrient recycling.

The production of biofuels from microalgae is associated with high demands of nutrients (nitrogen and phosphorus) required for growth. Recycling nutrients from the residual biomass is essential to obtain a sustainable production. In this work, the aqueous phase obtained from flash hydrolysis of Scenedesmus sp. was used as cultivation medium for a microalga of the same genus, to assess the feasibility of this technique for nutrient recycling purposes. Batch and continuous cultivations were carried out, to determine growth performances in this substrate compared to standard media, and verify if a stable biomass production could be obtained. In continuous experiments, the effect of hydrolysate inlet concentration and of residence time were assessed to optimize nutrient supply in relation to productivity. Results obtained show that nutrient recycling is feasible by treating biomass with flash hydrolysis, and Scenedesmus is capable of recycling large amounts of recovered nutrients.

Cultivation of Scenedesmus obliquus in photobioreactors: effects of light intensities and light-dark cycles on growth, productivity, and biochemical composition.

One of the main parameters influencing microalgae production is light, which provides energy to support metabolism but, if present in excess, can lead to oxidative stress and growth inhibition. In this work, the influence of illumination on Scenedesmus obliquus growth was assessed by cultivating cells at different light intensities in a flat plate photobioreactor. S. obliquus showed a maximum growth rate at 150 µmol photons m(-2) s(-1). Below this value, light was limiting for growth, while with more intense illumination photosaturation effects were observed, although cells still showed the ability to duplicate. Looking at the biochemical composition, light affected the pigment contents only while carbohydrate, lipid, and protein contents remained stable. By considering that in industrial photobioreactors microalgae cells are subjected to light-dark cycles due to mixing, algae were also grown under pulsed illumination (5, 10, and 15 Hz). Interestingly, the ability to exploit pulsed light with good efficiency required a pre-acclimation to the same conditions, suggesting the presence of a biological response to these conditions.

Adjusted light and dark cycles can optimize photosynthetic efficiency in algae growing in photobioreactors.

Biofuels from algae are highly interesting as renewable energy sources to replace, at least partially, fossil fuels, but great research efforts are still needed to optimize growth parameters to develop competitive large-scale cultivation systems. One factor with a seminal influence on productivity is light availability. Light energy fully supports algal growth, but it leads to oxidative stress if illumination is in excess. In this work, the influence of light intensity on the growth and lipid productivity of Nannochloropsis salina was investigated in a flat-bed photobioreactor designed to minimize cells self-shading. The influence of various light intensities was studied with both continuous illumination and alternation of light and dark cycles at various frequencies, which mimic illumination variations in a photobioreactor due to mixing. Results show that Nannochloropsis can efficiently exploit even very intense light, provided that dark cycles occur to allow for re-oxidation of the electron transporters of the photosynthetic apparatus. If alternation of light and dark is not optimal, algae undergo radiation damage and photosynthetic productivity is greatly reduced. Our results demonstrate that, in a photobioreactor for the cultivation of algae, optimizing mixing is essential in order to ensure that the algae exploit light energy efficiently.

Excess CO2 supply inhibits mixotrophic growth of Chlorella protothecoides and Nannochloropsis salina.

Mixotrophy can be exploited to support algal growth over night or in dark-zones of a photobioreactor. In order to achieve the maximal productivity, however, it is fundamental also to provide CO(2) in excess to maximize photosynthetic activity and phototropic biomass production. The aim of this paper is to verify the possibility of exploiting mixotrophy in combination with excess CO(2). Two species with high biomass productivity were selected, Nannochloropsis salina and Chlorella protothecoides. Different organic substrates available at industrial scale were tested, and glycerol chosen for its ability to support growth of both species. In mixotrophic conditions, excess CO(2) stimulated photosynthesis but blocked the metabolization of the organic substrate, thus canceling the advantages of mixotrophy. By cultivating microalgae under day-night cycle, organic substrate supported growth during the night, but only if CO(2) supply was not provided. This represents thus a possible method to reconcile CO(2) stimulation of photosynthesis with mixotrophy.