Couplage de la fermentation sombre et de l’hétérotrophie microalgale: influence du mélange de métabolites fermentaires, de la lumière, de la température et des bactéries fermentaires sur la croissance algale

Growing microalgae in heterotrophic mode present several advantages over autotrophic mode such as a higher productivity in terms of biomass and lipids for biofuels production. Nevertheless, this process is limited by the production cost associated with the organic substrate (i.e. glucose) and fermenters sterilization costs. Dark fermentation effluents, mainly composed of acetate and butyrate, could be used as a low-cost medium to grow microalgae heterotrophically or mixotrophically. The aims of this PhD were i) to optimize microalgae growth on various mixtures of fermentations metabolites using the presence or absence of light and different cultivation temperatures and ii) to assess the feasibility of using unsterilized fermentation effluents. First, a model based on mass balance was built to characterize heterotrophic growth rates and yields when Chlorella sorokiniana and Auxenochlorella protothecoides were supplemented with different mixtures of acetate and butyrate. Results showed that the acetate:butyrate ratio and the butyrate concentration per se were two key parameters for promoting heterotrophic growth. Then, further studies showed that the presence of light and the use of suboptimal temperature (30 °C) could reduce the butyrate inhibition on growth by either triggering autotrophic production of biomass or enhancing growth on acetate. Finally, it was shown that microalgae could outcompete fermentation bacteria for acetate when growing on raw dark fermentation effluents, thanks to a fast algal growth on acetate (1.75 d-1) and a drastic change of culture conditions to the detriment of bacterial growth.La production de microalgues en hétérotrophie présente plusieurs avantages pour la production de biocarburants par rapport à la production autotrophe, comme une productivité plus importante en termes de biomasse et de lipides. Cependant, le développement industriel de ce procédé est limité par les coûts de productions associés au substrat organique (i.e. glucose) et à ceux liés à la stérilisation des fermenteurs. Les effluents de fermentation sombre, composés principalement d’acétate et de butyrate, pourraient être utilisés comme milieux de culture peu onéreux pour la culture hétérotrophe ou mixotrophe de microalgues. Les objectifs de cette thèse étaient i) de mieux appréhender la croissance algale sur des mélanges variés d’acétate et de butyrate en fonction de la présence ou l’absence de lumière et de la température de croissance et ii) d’évaluer la faisabilité d’utiliser des effluents de fermentation non stérilisés pour soutenir la croissance de microalgues oléagineuses. Tout d’abord, un modèle basé sur des bilans de masse a été construit afin de caractériser (taux de croissance et rendements) la croissance hétérotrophe de Chlorella sorokiniana et Auxenochlorella protothecoides sur des mélanges d’acétate et de butyrate. Les résultats ont montré que le rapport acétate:butyrate et la concentration en butyrate étaient deux paramètres clés pour soutenir la croissance hétérotrophe. Puis, il a été démontré que la présence de lumière et l’utilisation d’une température suboptimale (30 °C) pour la croissance algale permettaient de réduire l’inhibition du butyrate en permettant une production de biomasse autotrophe ou en améliorant la croissance sur acétate. Enfin, il a été montré que les microalgues peuvent être compétitives sur l’acétate lors de la croissance sur des effluents bruts de fermentation sombre en présence de bactéries fermentaires, grâce à la croissance rapide des microalgues sur acétate (1.75 j-1) et à un changement drastique des conditions de culture peu favorables à la croissance des bactéries d’origine fermentaire

Dynamic metabolic modeling of heterotrophic and mixotrophic microalgal growth on fermentative wastes

Microalgae are promising microorganisms for the production of numerous molecules of interest, such as pigments, proteins or triglycerides that can be turned into biofuels. Heterotrophicor mixotrophic growth on fermentative wastes represents an interesting approach to achieving higher biomass concentrations, while reducing cost and improving the environmental footprint. Fermentative wastes generally consist of a blend of diverse molecules and it is thus crucial to understand micro algal metabolism in such conditions, where switching between substrates might occur. Metabolic modeling has proven to be an efficient tool for understanding metabolism and guiding the optimization of biomass or target molecule production. Here, we focused on the metabolism of Chlorella sorokiniana growing heterotrophically and mixotrophically on acetate and butyrate. The metabolism was represented by 172metabolic reactions. The DRUM modeling framework with a mildly relaxed quasi-steady state assumption was used to account for the switching between substrates and the presence of light. Nine experiments were used to calibrate the model and nine experiments for the validation. The model efficiently predicted the experimental data, including the transient behavior during heterotrophic, autotrophic, mixotrophic and diauxic growth. It shows that anaccurate model of metabolism can now be constructed, even in dynamic conditions, with the presence of several carbon substrates. It also opens new perspectives for the heterotrophicand mixotrophic use of microalgae, especially for biofuel production from wastes

Dynamic metabolic modeling of heterotrophic and mixotrophic microalgal growth on fermentative wastes.

Microalgae are promising microorganisms for the production of numerous molecules of interest, such as pigments, proteins or triglycerides that can be turned into biofuels. Heterotrophic or mixotrophic growth on fermentative wastes represents an interesting approach to achieving higher biomass concentrations, while reducing cost and improving the environmental footprint. Fermentative wastes generally consist of a blend of diverse molecules and it is thus crucial to understand microalgal metabolism in such conditions, where switching between substrates might occur. Metabolic modeling has proven to be an efficient tool for understanding metabolism and guiding the optimization of biomass or target molecule production. Here, we focused on the metabolism of Chlorella sorokiniana growing heterotrophically and mixotrophically on acetate and butyrate. The metabolism was represented by 172 metabolic reactions. The DRUM modeling framework with a mildly relaxed quasi-steady-state assumption was used to account for the switching between substrates and the presence of light. Nine experiments were used to calibrate the model and nine experiments for the validation. The model efficiently predicted the experimental data, including the transient behavior during heterotrophic, autotrophic, mixotrophic and diauxic growth. It shows that an accurate model of metabolism can now be constructed, even in dynamic conditions, with the presence of several carbon substrates. It also opens new perspectives for the heterotrophic and mixotrophic use of microalgae, especially for biofuel production from wastes

Potentialities of dark fermentation effluent as substrates for microalgae growth: A review

In recent years, coupling bacterial dark fermentation (DF) and heterotrophic cultivation of microalgae (HCM) has been pointed out as a promising sustainable approach for producing both gaseous and liquid biofuels. Complex organic waste and effluents that are not susceptible to be directly degraded by microalgae are first converted into volatile fatty acids (VFAs) and hydrogen by DF. In this work, the feasibility of using DF effluents to sustain has been thoroughly reviewed and evaluated. Promising perspectives in terms of microalgae biomass and lipids production are proposed and can be extended as guidelines to promote HCM whatever the organic waste used. Abiotic and biotic factors from DF effluents that promote or inhibit microalgae growth are discussed as well as the use of unsterile DF effluents. Overall, the microalgae growth is favored on effluents containing high acetate concentration (> 3 g L−1), with a high acetate:butyrate ratio (> 2.5), and when pH is strictly controlled. At a low acetate:butyrate ratio (<1) and/or high total metabolites concentrations (>10 g L−1), a low substrate:microalgae ratio and the presence of light appear to enhance microalgae growth. Butyrate content appears to be a key factor when coupling DF/HCM since high butyrate concentration inhibits the microalgae growth

Replacing incandescent lamps with an LED panel for hydrogen production by photofermentation: Visible and NIR wavelength requirements

International audienceHydrogen production by Rhodobacter capsulatus is a photobiological anaerobic process requiring light as energy source. In this study, the influence of visible and near-infrared (NIR) parts of light spectra from incandescent lamp and LED panels on hydrogen production was investigated. The results showed that the lack of the visible part of the incandescent lamp light spectrum (17% of the lamp light intensity) reduced hydrogen production by 50%. NIR wavelength only partially sustained photofermentation due to light limitation reached at low bacterial concentration. Hydrogen production with NIR light source was only 58% of hydrogen obtained with an incandescent lamp used at the same irradiance. To maximize hydrogen production and flow rate, visible and NIR wavelength should be used concomitantly as light source. Using an energy-efficient LED panel with light spectrum designed to promote photofermentation, hydrogen production and flow rate were equivalent to the ones reached with incandescent lamp as light source

Growth of Chlorella sorokiniana on a mixture of volatile fatty acids: The effects of light and temperature

International audienceThis study investigated the influence of light and temperature on Chlorella sorokiniana grown on a mixture of acetate and butyrate, two of the volatile fatty acids produced by dark fermentation. Exposure to light caused autotrophic biomass production (56% of the final biomass) and reduced the time to reach butyrate exhaustion to 7 days at 25°C from 10 days in the dark. For growth on acetate at the optimum temperature (35°C), the presence of butyrate reduced the growth rate (by 46%) and the carbon yield (by 36%). For successful microalgae growth on dark fermentation effluent, butyrate inhibition may be reduced by setting the temperature to 30°C and providing light

Potentialities of dark fermentation effluent as substrates for microalgae growth: A review

In recent years, coupling bacterial dark fermentation (DF) and heterotrophic cultivation of microalgae (HCM) has been pointed out as a promising sustainable approach for producing both gaseous and liquid biofuels. Complex organic waste and effluents that are not susceptible to be directly degraded by microalgae are first converted into volatile fatty acids (VFAs) and hydrogen by DF. In this work, the feasibility of using DF effluents to sustain has been thoroughly reviewed and evaluated. Promising perspectives in terms of microalgae biomass and lipids production are proposed and can be extended as guidelines to promote HCM whatever the organic waste used. Abiotic and biotic factors from DF effluents that promote or inhibit microalgae growth are discussed as well as the use of unsterile DF effluents. Overall, the microalgae growth is favored on effluents containing high acetate concentration (> 3 g L−1), with a high acetate:butyrate ratio (> 2.5), and when pH is strictly controlled. At a low acetate:butyrate ratio (<1) and/or high total metabolites concentrations (>10 g L−1), a low substrate:microalgae ratio and the presence of light appear to enhance microalgae growth. Butyrate content appears to be a key factor when coupling DF/HCM since high butyrate concentration inhibits the microalgae growth

Comparison between the model and experimental data for <i>Chlorella sorokiniana</i> mixotrophic and autotrophic growth.

<p>Simulations are represented by full lines (conditions used for calibration). Experimental results are represented by large dots, diamonds or triangles. Red: acetate; blue: butyrate; yellow: biomass. A. Autotrophic growth. B. Mixotrophic growth with 0.3 gC.L^-1 acetate C. Mixotrophic growth with 0.3 gC.L^-1 butyrate. D. Mixotrophic growth with 0.3 gC.L^-1 acetate and 0.3 gC.L^-1 butyrate. Only one of the experimental triplicates is represented here. The simulations for all triplicates are available in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005590#pcbi.1005590.s011" target="_blank">S7 Fig</a>.</p

Definition and reduction of sub-networks formed from metabolic reactions of <i>Chlorella sorokiniana</i> for heterotrophic and mixotrophic growth.

<p>Definition and reduction of sub-networks formed from metabolic reactions of <i>Chlorella sorokiniana</i> for heterotrophic and mixotrophic growth.</p

Comparison between the model and experimental data for <i>Chlorella sorokiniana</i> heterotrophic growth mixtures of acetate and butyrate.

<p>Simulations are represented by full lines (conditions used for calibration) or dashed lines (conditions used for validation). Experimental results are represented by large dots, triangles or diamonds. Red: acetate (gC.L^-1); blue: butyrate (gC.L^-1); yellow: biomass (g.L^-1). A. Growth on 0.25 gC.L^-1 acetate and 0.25 gC.L^-1 butyrate. B. Growth on 0.25 gC.L^-1 acetate and 0.5 gC.L^-1 butyrate. C. Growth on 0.4 gC.L^-1 acetate and 0.1 gC.L^-1 butyrate. D. Growth on 0.5 gC.L^-1 acetate and 0.9 gC.L^-1 butyrate. E. Growth on 0.9 gC.L^-1 acetate and 0.1 gC.L^-1 butyrate. Only one of the experimental triplicates is represented here. The simulations for all triplicates are available in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005590#pcbi.1005590.s009" target="_blank">S5 Fig</a>.</p