Comparison of photobioreactors and optimal light regime for rapid vegetative propagation of transgenic Undaria pinnatifida gametophytes

BACKGROUND: Previously, tachyplesin gene (tac) has been successfully transferred into Undaria pinnatifida gametophytes using the method of microprojectile bombardment transformation. The objectives of this study were to compare and evaluate the performance of bubble-column and airlift bioreactors to determine a preferred configuration of bioreactor for vegetative propagation of transgenic U. pinnatifida gametophytes, and to then investigate the influence of light on vegetative propagation of these gametophytes, including incident light intensity, photoperiod and light quality to resolve the problems of rapid vegetative propagation within the selected bioreactor. RESULTS: Experimental results showed that final dry cell density in the airlift bioreactor was 12.7% higher than that in the bubble-column bioreactor under the optimal aeration rate of 1.2 L air min(-1) L-1 culture. And a maximum final dry cell density of 2830 mg L-1 was obtained within the airlift bioreactor using blue light at 40 mu mol m(-2) s(-1) with a light/dark cycle of 14/10 (h). Polymerase chain reaction (PCR) analysis indicated that genes (bar and tac) were not lost during rapid vegetative propagation within the airlift bioreactor. CONCLUSION: The airlift bioreactor was shown to be much more suitable for rapid vegetative propagation of transgenic U. pinnatifida gametophytes than the bubble-column bioreactor in the laboratory. The use of blue light allows improvement of vegetative propagation of transgenic U. pinnatifida gametophytes. (C) 2009 Society of Chemical Industry

Comparison of photobioreactors for cultivation of Undaria pinnatifida gametophytes

Undaria pinnatifida gametophytes were grown in 2.5 l bubble column and airlift reactor at 25 degreesC and light intensity of 40 mumol m(-2) s(-1) for 6 days. With aeration at 1 l min(-1), the airlift reactor yielded higher growth rate (0.12 mg DW ml(-1) d(-1)) than a bubble column (0.08 mg DW ml(-1) d(-1)). The advantages were related to the more homogeneous fluid dynamic characteristics of the airlift reactor.

Modeling, characterization and microalgal cultivation in an airlift panel photobioreactors.

Microalgae cultures are attracting great attentions in many industrial applications. However, one of the technical challenges is to cut down the capital and operational costs of microalgae production systems, with special difficulty in reactor design and scale-up. The thesis work open with an overview on the microalgae cultures as a possible answer to solve some of the upcoming planet issues and their applications in several fields. After the work offers a general outline on the state of the art of microalgae culture systems, taking a special look to the enclosed photobioreactors (PBRs). The overall objective of this study is to advance the knowledge of PBRs design and lead to innovative large scale processes of microalgae cultivation. An airlift flat panel photobioreactor was designed, modeled and experimentally characterized. The gas holdup, liquid flow velocity and oxygen mass transfer of the reactor were experimentally determined and mathematically modeled, and the performance of the reactor was tested by cultivation of microalgae. The model predicted data correlated well with experimental data, and the high concentration of suspension cell culture could be achieved with controlled conditions. The reactor was inoculated with the algal strain Scenedesmus obliquus sp. first and with Chlorella sp. later and sparged with air. The reactor was operated in batch mode and daily monitored for pH, temperature, and biomass concentration and activity. The productivity of the novel device was determined, suggesting the proposed design can be effectively and economically used in carbon dioxide mitigation technologies and in the production of algal biomass for biofuel and other bioproducts. Those research results favored the possibility of scaling the reactor up into industrial scales based on the models employed, and the potential advantages and disadvantages were discussed for this novel industrial design.

Outdoor production of Isochrysis galbana (T-iso) in industrial scale photobioreactors and modelling of its photosynthesis and respiration rate

Isochrysis galbana is a widely-used strain in aquaculture in spite of its low productivity. To maximize the productivity of processes based on this microalgae strain, a model was developed considering the influence of irradiance, temperature, pH and dissolved oxygen concentration on the photosynthesis and respiration rate. Results demonstrate that this strain tolerates temperatures up to 35ºC but it is highly sensitive to irradiances higher than 500 µE·m-2·s-1 and dissolved oxygen concentrations higher than 11 mg·l-1. With the researcher group of the “Universidad de Almeria”, the developed model was validated using data from an industrial-scale outdoor tubular photobioreactor demonstrating that inadequate temperature and dissolved oxygen concentrations reduce productivity to half that which is maximal, according to light availability under real outdoor conditions. The developed model is a useful tool for managing working processes, especially in the development of new processes based on this strain and to take decisions regarding optimal control strategies. Also the outdoor production of Isochrysis galbana T-iso in industrial size tubular photobioreactors (3.0 m3) has been studied. Experiments were performed modifying the dilution rate and evaluating the biomass productivity and quality, in addition to the overall performance of the system. Results confirmed that T-iso can be produced outdoor at commercial scale in continuous mode, productivities up to 20 g·m-2·day-1 of biomass rich in proteins (45%) and lipids (25%) being obtained. The utilization of this type of photobioreactors allows controlling the contamination and pH of the cultures, but daily variation of solar radiation imposes the existence of inadequate dissolved oxygen concentration and temperature at which the cells are exposed to inside the reactor. Excessive dissolved oxygen reduced the biomass productivity to 68% of maximal, whereas inadequate temperature reduces to 63% of maximal. Thus, optimally controlling these parameters the biomass productivity can be duplicated. These results confirm the potential to produce this valuable strain at commercial scale in optimally designed/operated tubular photobioreactors as a biotechnological industry.

ESTUDO DA CONCENTRAÇÃO DE BIOMASSSA DE MICROALGAS POR FILTRAÇÃO E FLOCULAÇÃO

O cultivo de microalgas é uma matéria prima para produção de biocombustível e de captura de carbono devido a vantagens como alta produção de biomassa e rápido crescimento quando comparado com outras fontes de energia e não necessitar de terra fértil. O presente trabalho teve como objetivo estudar métodos de concentração da biomassa. A microalga utilizada foi a Isochrysis galbana. Os cultivos tiveram duração de 20 dias e concentração inicial de 7.104 cel/mL no meio de cultivo F2/Guillard. e foram realizados em fotobioreatores de 500 mL, 3 L e 12 L. Os experimentos foram conduzidos em foto-período de 12 h claro/escuro, com temperatura de 27 a 29 C. Ao final dos cultivos, as amostras foram levadas para a sequência de processos de separação. Inicialmente, foram realizados ensaios de microfiltração em membrana com porosidade de 0,45 m em procedimento do tipo dead-end e constatou-se a rápida e intensa formação de camada de fouling. Acrescentou-se uma etapa de separação por floculação preliminar à microfiltração, utilizando-se Al2(SO4)3 como agente floculante. O meio coagulado foi então filtrado e microfiltrado. O estudo combinado das 3 etapas de separação possibilitou 99% de remoção de biomassa.O teor de óleo obtido foi de 22,4%. Portanto, o trabalho apresenta uma configuração de concentração da biomassa Isochrysis galbana visando o processo de produção de biocombustíveis

The CO2 microalgae biorefinery: high value products and biofuels using halophilic microalgae in the “D-Factory”

Fuel-only algal systems are not economically feasible because yields are too low and costs too high for producing microalgal biomass compared to using agricultural residues e.g. straw. Biorefineries which integrate biomass conversion processes and equipment to produce fuels, power and chemicals from biomass, offer a solution. The CO2 microalgae biorefinery (D-Factory) is a 10 million Euro FP7-funded project which will cultivate the microalga Dunaliella in highly saline non-potable waters in photobioreactors and open raceways and apply biorefinery concepts and European innovations in biomass processing technologies to develop a basket of compounds from Dunaliella biomass, including the high value nutraceutical, β-carotene, and glycerol. Glycerol now finds markets both as a green chemical intermediate and as a biofuel in CHP applications as a result of novel combustion technology. Driving down costs by recovering the entire biomass of Dunaliella cells from saline cultivation water poses one of the many challenges for the D-Factory because Dunaliella cells are both motile, and do not possess an external cell wall, making them highly susceptible to cell rupture. Controlling expression of desired metabolic pathways to deliver the desired portfolio of compounds flexibly and sustainably to meet market demand is another. The first prototype D-Factory in Europe will be operational in 48 months, and will serve as a robust manifestation of the business case for global investment in algae biorefineries and in large-scale production of microalgae.

Cultivo e avaliação do potencial da microalga Chlorella zofingiensis para a produção de biodiesel e produtos de valor acrescentado

O presente trabalho tem como objetivo o cultivo da microalga Chlorella zofingiensis, e a avaliação da sua potencial aplicação na produção de biodiesel e de produtos de valor acrescentado, de entre os quais se destacam os antioxidantes. Com o intuito da produção de biocombustível é necessário efetuar o cultivo da microalga num volume que permita a obtenção de elevada quantidade de biomassa para a concretização do trabalho. Além deste biocombustível, existe ainda a possibilidade de valorização de alguns produtos com valor comercial, como é o caso da astaxantina, a saber na área farmacêutica, alimentar ou até mesmo cosmética. O cultivo da microalga foi feito em meio Bold’s Basal Medium (BBM), inicialmente em matrazes de 5 L e, quando se obteve uma cultura suficientemente densa, inocularam-se fotobiorreatores de 50 L. Conseguiu-se atingir uma concentração máxima de 0,76 g/L, no reator de 5 L, após cerca de 6 semanas de ensaio. Por sua vez, em fotobiorreatores de 50 L, a concentração máxima obtida foi de 0,4 g/L, após 4 semanas de ensaio. Nestas culturas foi possível obter-se uma percentagem lipídica de 7 %, apresentado concentração de pigmentos por litro de cultura na ordem dos 10 mg/L, 4 mg/L e 2 mg/L de clorofila a, clorofila b e carotenoides totais, respetivamente. Com esta percentagem lipídica recuperaram-se 400 mg de óleo, obtendo-se posteriormente 280 mg de biodiesel. Pela análise à amostra de biodiesel obtida foi possível obter o perfil lipídico desta microalga, quando cultivada em meio BBM, sendo 41% de ácido palmítico (C16:0), 9% de ácido esteárico (C18:0), 27% de ácido oleico (C18:1) e 23% de ácido linoleico (C18:2). Os resultados obtidos mostram que a Chlorella zofingiensis é uma microalga com interesse potencial para a produção de clorofila e carotenóides, mas não para o óleo para a produção de biodiesel.

Use of swine waste-derived biogas to enhance microalgae productivity.

The effects of swine wastewater-derived biogas on microalgae productivity were determined. Experiments were conducted in a closed photobioreactor containing digestate effluent as culturing media and biogas in the headspaceas source of CO2. Experiments were carried out under mixothrophic and autothrophic conditions. Results showed that autotrophic growth rate (0.6 d-1)was twofoldfaster than mixotrophic. Frequent reinjections of biogas containing up to 2,000 ppm of hydrogen sulfide was not inhibitory to microalgae growth. The rapid removal of H2S in the system suggests photobioreactors can be an interesting alternative to biogas purification. A model to estimate microalgae productivity based on the amount of available CO2, inorganic and organic carbon was developedand showed good data fit correlation (r²= 0.99).

Determination of microalgae cellular composition after phycoremediation of swine wastewaters.

Phycoremediation of swine wastewaters has been widely reported as an attractive tertiary treatment system, that effectively removes the excessive nutrient loadswhilst offering a valuable source of feedstock biomass. Digestate from an upflow anaerobic sludge blanket (UASB, 6%v/v) and a nitrification reactor (NR; 50% v/v) were used as culturing media to microalgae. Experiments were carried out in lab scale photobioreactors (PBRs) using a consortia of Chlorella and Scenedesmus. Ammonia (44 to 90%) and phosphorus (77%) were efficiently removed from both effluents tested after 4 days. Microalgae biomass harvested from the UASB effluent showed 57, 34 and 1% of proteins, carbohydrates and lipids, respectively. Comparatively, the cellular composition of microalgae grown on NR effluent had lower protein (43%) but higher carbohydrate (42%) contents. Negligible difference in lipid fraction was observed independently of the effluents tested. The results suggest that the biomass harvested from phycoremediation of swine wastewaters can offer a valuable protein and carbohydrate feedstock for nutritional and biotechnological applications.

Improvements in Fermentative Hydrogen Production through Physiological Manipulation and Metabolic Engineering

La production biologique d'hydrogène (H2) représente une technologie possible pour la production à grande échelle durable de H2 nécessaire pour l'économie future de l'hydrogène. Cependant, l'obstacle majeur à l'élaboration d'un processus pratique a été la faiblesse des rendements qui sont obtenus, généralement autour de 25%, bien en sous des rendements pouvant être atteints pour la production de biocarburants à partir d'autres processus. L'objectif de cette thèse était de tenter d'améliorer la production d'H2 par la manipulation physiologique et le génie métabolique. Une hypothèse qui a été étudiée était que la production d'H2 pourrait être améliorée et rendue plus économique en utilisant un procédé de fermentation microaérobie sombre car cela pourrait fournir la puissance supplémentaire nécessaire pour une conversion plus complète du substrat et donc une production plus grande d'H2 sans l'aide de l'énergie lumineuse. Les concentrations optimales d’O2 pour la production de H2 microaérobie ont été examinées ainsi que l'impact des sources de carbone et d'azote sur le processus. La recherche présentée ici a démontré la capacité de Rhodobacter capsulatus JP91 hup- (un mutant déficient d’absorption-hydrogénase) de produire de l'H2 sous condition microaérobie sombre avec une limitation dans des quantités d’O2 et d'azote fixé. D'autres travaux devraient être entrepris pour augmenter les rendements d'H2 en utilisant cette technologie. De plus, un processus de photofermentation a été créé pour améliorer le rendement d’H2 à partir du glucose à l'aide de R. capsulatus JP91 hup- soit en mode non renouvelé (batch) et / ou en conditions de culture en continu. Certains défis techniques ont été surmontés en mettant en place des conditions adéquates de fonctionnement pour un rendement accru d'H2. Un rendement maximal de 3,3 mols de H2/ mol de glucose a été trouvé pour les cultures en batch tandis que pour les cultures en continu, il était de 10,3 mols H2/ mol de glucose, beaucoup plus élevé que celui rapporté antérieurement et proche de la valeur maximale théorique de 12 mols H2/ mol de glucose. Dans les cultures en batch l'efficacité maximale de conversion d’énergie lumineuse était de 0,7% alors qu'elle était de 1,34% dans les cultures en continu avec un rendement de conversion maximum de la valeur de chauffage du glucose de 91,14%. Diverses autres approches pour l'augmentation des rendements des processus de photofermentation sont proposées. Les résultats globaux indiquent qu'un processus photofermentatif efficace de production d'H2 à partir du glucose en une seule étape avec des cultures en continu dans des photobioréacteurs pourrait être développé ce qui serait un processus beaucoup plus prometteur que les processus en deux étapes ou avec les co-cultures étudiés antérieurément. En outre, l'expression hétérologue d’hydrogenase a été utilisée comme une stratégie d'ingénierie métabolique afin d'améliorer la production d'H2 par fermentation. La capacité d'exprimer une hydrogénase d'une espèce avec des gènes de maturation d'une autre espèce a été examinée. Une stratégie a démontré que la protéine HydA orpheline de R. rubrum est fonctionnelle et active lorsque co-exprimée chez Escherichia coli avec HydE, HydF et HydG provenant d'organisme différent. La co-expression des gènes [FeFe]-hydrogénase structurels et de maturation dans des micro-organismes qui n'ont pas une [FeFe]-hydrogénase indigène peut entraîner le succès dans l'assemblage et la biosynthèse d'hydrogénase active. Toutefois, d'autres facteurs peuvent être nécessaires pour obtenir des rendements considérablement augmentés en protéines ainsi que l'activité spécifique des hydrogénases recombinantes. Une autre stratégie a consisté à surexprimer une [FeFe]-hydrogénase très active dans une souche hôte de E. coli. L'expression d'une hydrogénase qui peut interagir directement avec le NADPH est souhaitable car cela, plutôt que de la ferrédoxine réduite, est naturellement produit par le métabolisme. Toutefois, la maturation de ce type d'hydrogénase chez E. coli n'a pas été rapportée auparavant. L'opéron hnd (hndA, B, C, D) de Desulfovibrio fructosovorans code pour une [FeFe]-hydrogénase NADP-dépendante, a été exprimé dans différentes souches d’E. coli avec les gènes de maturation hydE, hydF et hydG de Clostridium acetobutylicum. L'activité de l'hydrogénase a été détectée in vitro, donc une NADP-dépendante [FeFe]-hydrogénase multimérique active a été exprimée avec succès chez E. coli pour la première fois. Les recherches futures pourraient conduire à l'expression de cette enzyme chez les souches de E. coli qui produisent plus de NADPH, ouvrant la voie à une augmentation des rendements d'hydrogène via la voie des pentoses phosphates.

Algal Biofuels: Challenges and Opportunities

Biodiesel production using microalgae is attractive in a number of respects. Here a number of pros and cons to using microalgae for biofuels production are reviewed. Algal cultivation can be carried out using non-arable land and non-potable water with simple nutrient supply. In addition, algal biomass productivities are much higher than those of vascular plants and the extractable content of lipids that can be usefully converted to biodiesel, triacylglycerols (TAGs) can be much higher than that of the oil seeds now used for first generation biodiesel. On the other hand, practical, cost-effective production of biofuels from microalgae requires that a number of obstacles be overcome. These include the development of low-cost, effective growth systems, efficient and energy saving harvesting techniques, and methods for oil extraction and conversion that are environmentally benign and cost-effective. Promising recent advances in these areas are highlighted.

Kinetic and thermodynamic investigation of Arthrospira (Spirulina) platensis fed-batch cultivation in a tubular photobioreactor using urea as nitrogen source

BACKGROUND: Fed-batch culture allows the cultivation of Arthrospira platensis using urea as nitrogen source. Tubular photobioreactors substantially increase cell growth, but the successful use of this cheap nitrogen source requires a knowledge of the kinetic and thermodynamic parameters of the process. This work aims at identifying the effect of two independent variables, temperature (T) and urea daily molar flow-rate (U), on cell growth, biomass composition and thermodynamic parameters involved in this photosynthetic cultivation. RESULTS: The optimal values obtained were T = 32 degrees C and U = 1.16 mmol L-1 d-1, under which the maximum cell concentration was 4186 +/- 39 mg L-1, cell productivity 541 +/- 5 mg L-1 d-1 and yield of biomass on nitrogen 14.3 +/- 0.1 mg mg-1. Applying an Arrhenius-type approach, the thermodynamic parameters of growth (?H* = 98.2 kJ mol-1; ?S* = - 0.020 kJ mol-1 K-1; ?G* = 104.1 kJ mol-1) and its thermal inactivation (Delta H-D(0) =168.9 kJ mol-1; Delta S-D(0) = 0.459 kJ mol-1 K-1; Delta G(D)(0) =31.98 kJ mol-1) were estimated. CONCLUSIONS: To maximize cell growth T and U were simultaneously optimized. Biomass lipid content was not influenced by the experimental conditions, while protein content was dependent on both independent variables. Using urea as nitrogen source prevented the inhibitory effect already observed with ammonium salts. Copyright (c) 2012 Society of Chemical Industry

Effects of photobioreactor configuration, nitrogen source and light intensity on the fed-batch cultivation of Arthrospira (Spirulina) platensis. Bioenergetic aspects

Bioenergetic analysis may be applied in order to predict microbial growth yields, based on the Gibbs energy dissipation and mass conservation principles of the overall growth reaction. The bioenergetics of the photoautotrophic growth of the cyanobacterium Arthrospira (Spirulina) platensis was investigated in different bioreactor configurations (tubular photobioreactor and open ponds) using different nitrogen sources (nitrate and urea) and under different light intensity conditions to determine the best growing conditions in terms of Gibbs energy dissipation, number of photons to sustain cell growth and phototrophic energy yields distribution in relation to the ATP and NADPH formation, and release of heat. Although an increase in the light intensity increased the Gibbs energy dissipated for cell growth and maintenance with both nitrogen sources, it did not exert any appreciable influence on the moles of photons absorbed by the system to produce one C-mol biomass. On the other hand, both bioenergetic parameters were higher in cultures with nitrate than with urea, likely because of the higher energy requirements needed to reduce the former nitrogen source to ammonia. They appreciably increased also when open ponds were substituted by the tubular photobioreactor, where a more efficient light distribution ensured a remarkably higher cell mass concentration. The estimated percentages of the energy absorbed by the cell showed that, compared with nitrate, the use of urea as nitrogen source allowed the system to address higher energy fractions to ATP production and light fixation by the photosynthetic apparatus, as well as a lower fraction released as heat. The best energy yields values on Gibbs energy necessary for cell growth and maintenance were achieved in up to 4-5 days of cultivation, indicating that it would be the optimum range to maintain cell growth. Thanks to this better bioenergetic situation, urea appears to be a quite promising low-cost, alternative nitrogen source for Arthrospira platensis cultures in photobioreactors. (C) 2011 Elsevier Ltd. All rights reserved.

Biodiesel renovável derivado de microalgas: avanços e perspectivas tecnológicas

Microalgae are a promising source of raw material for biodiesel production. This review discusses the latest developments related to the application of microalgae biomass for biodiesel production. Characterization of fatty acid of microalgae and comparisons with other sources of raw materials and processes are presented. Furthermore, technological perspectives and approaches for growing microalgae in photobioreactors, microalgal oil extraction techniques, and procedures for synthesizing biodiesel are reviewed.

An Investigation On The Use Of Microalgae For Biogas Enrichment

There is a need for biomethane capture and carbon dioxide sequestration to mitigate evident global climate change. This research work investigated the potential for microalgae to remove CO2 from biogas as a biotechnical method for upgrading the thermal value for subsequent compression, liquification, or introduction to natural gas pipelines. Because biogas is largely methane, the effect of high methane environments on mixed microalgae was explored and found that specific carbon utilization rates were not statistically different when microalgae were exposed to biogas environments (70% v/v CH4) , relative to high CO2 environment. The uses of conventional bubbled column photobioreactors (PBR) were assessed for CO2 removal and subsequent CH4 enrichment. A continuously-bubbled biogas PBR (cB-PBR5) and intermittently-bubbled biogas PBR (iB-PBR) experienced CO2 loading rates of about 1664 and 832 mg C/L*day and showed 30.0 and 60.1 % carbon removal, respectively. However, a lack of biogas enrichment and issues associated growth inhibition due to high CO2 environments as well as stripping the dissolved gases, namely oxygen and nitrogen, from the bulk liquid and introduction to the outlet gas prompted the consideration for gas/liquid separation using nonporous hollow-fiber (HF) membranes for CO2 transfer. The potential for two non-porous HF membrane materials [polydimethylsiloxane (PDMS) and composite polyurethane (PU)] were modeled along fiber length using a mechanistic model based on polymeric material transport properties (Gilmore et al., 2009). Based on a high CO2:CH4 permeability selectivity for PU of 76.2 the model predicted gas enrichment along an 8.5 cm fiber length. Because PDMS permeability selectivity is low (3.5), evident gas transfer was not predicated along a 34.3 cm length. Both of these HF materials were implemented in hollow-fiber membrane-carbonated biofilm (HFMcB) PBRs for microalgal-mediated biogas enrichment. Phototrophic biofilm colonization occurred on the membrane, where CO2 concentration was greatest. The presence of a biofilm demonstrated greater resiliency to high CO2 environments, compared to the conventional PBRs. However, as the PDMS model predicted, the PDMS HFMcBs did not demonstrate gas enrichment. These reactors received CO2 loading rates of 200 mg C/L*day based on PDMS permeability flux and showed approximately 65% removal of the total C transferred across the membrane. Thus, the HFMcBs demonstrated controlled carbonation of the bulk liquid via a nonporous HF membrane. Likewise, the experimental PU HFMcB did not show gas enrichment yet this result should be further explored due to the high permeability selectivity of the polymeric material. Chemical stratifications, namely pH and dissolved O2, present in a PDMS membrane-carbonated biofilm were analyzed using electrochemical microsensors. Results indicated that high DO (20 mg L-1) exists at surface of the biofilm where light availability is greatest and low pH microenvironments (pH=5.40) exist deep in the biofilm where the diffusive flux of CO2 drives transfer through the biofilm. The presence of a 400-600 ¿m liquid phase boundary layer was evident from microsensor profiles. Cryosectioning of the biofilm samples showed the biofilm to be approximately 1.17 ± 0.07 mm thick, suggesting that the high localized concentration of biomass associated with the phototrophic biofilm aided in overcoming inhibition in a microenvironment dominated by CO2(aq). Challenges of biofilm detachment and PBR fouling as well as microalgal growth inhibition in the presence of high CO2 content remain for applications of microalgae for biogas enrichment.