Phototrophic Cultivation Research Articles

Control of chitin nanofiber production by the lipid-producing diatom Cyclotella Sp. through fed-batch addition of dissolved silicon and nitrate in a bubble-column photobioreactor.

Diatoms are single-celled algae that make cell walls of nanopatterned biogenic silica called frustules through metabolic uptake of dissolved silicon and its templated condensation into biosilica. The centric marine diatom Cyclotella sp. also produces intracellular lipids and the valued coproduct chitin, an N-acetyl glucosamine biopolymer that is extruded from selected frustule pores as pure nanofibers. The goal of this study was to develop a nutrient feeding strategy to control the production of chitin nanofibers from Cyclotella with the coproduction of biofuel lipids. A two-stage phototrophic cultivation process was developed where Stage I set the cell suspension to a silicon-starved state under batch operation, and Stage II continuously added silicon and nitrate to the silicon-starved cells to enable one more cell doubling to 4 × 106 cellsmL-1 . The silicon delivery rate was set to enable a silicon-limited cell division rate under cumulative delivery of 0.8 mM Si and 1.2 mM nitrate (1.5:1 mol N/mol Si) over a 4- to 14-day addition period. In Stage II, both cell number and chitin production were linear with time. Cell number and the specific chitin production rate increased linearly with increasing silicon delivery rate to achieve cumulative product yields of 13 ± 1 mg chitin/109 cells and 33 ± 3 mg lipid/109 cells. Therefore, chitin production is controlled through cell division, which is externally controlled through silicon delivery. Lipid production was not linearly correlated to silicon delivery and occurred primarily during Stage I, just after the complete co-consumption of both dissolved silicon and nitrate. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:407-415, 2017.

New bioprocess tool for phototrophic cultivation.

New bioprocess tool for phototrophic cultivation.

Reverse osmosis concentrate conditioning for microalgae cultivation and a generalized workflow

Previous studies have demonstrated the feasibility of using wastewater as the nutrient source for microalgae cultivation. However, there is still a lack of guidance in wastewater conditioning to achieve optimum microalgae growth rate and complete N and P consumption in wastewater. In this study, a reverse osmosis concentrate (ROC) from the wastewater treatment unit of a dyeing company had been evaluated and conditioned for the phototrophic cultivation of Chlorella sp., with its toxicity, nutrient profile, salinity, pH and N/P ratio adjusted where necessary. After the wastewater was conditioned, Chlorella sp., which could not grow in the original ROC, achieved a growth rate comparable to that in an artificial medium. The percentages of N and P consumption were also balanced so that the nutrient utilization efficiency could be maximized. A generalized workflow and guidelines to develop wastewater conditioning plans for microalgae cultivation have also been proposed to guide the conditioning of wastewater streams for microalgae cultivation.

Optimization and mass culture of Acutodesmus obliquus RDS01 under open phototrophic pond cultivation for enhancing biodiesel production

ABSTRACTIn the optimization of medium for open phototrophic pond cultivation of microalgae, Acutodesmus obliquus RDS01, response surface methodology (RSM) was used and lipid, biomass and biodiesel production were analyzed. The experimental investigation showed that a good achievement and regression coefficient was determined highly (R2 = 0.999) in analysis of variance (ANOVA). The effect of six different macro-elements (NaNO3, KH2PO4, K2HPO4, MgSO4, CaCl2 and NaCl) on growth of Acutodesmus obliquus RDS01 was premeditated in bold basal medium (BBM). The absolute concentration of lipid was 220.07 mg mL−1, biomass 14.49 g L−1 and biodiesel 3.9 mL g−1. The results achieved in phototrophic open pond cultivation using optimized medium were approximately 2 times higher than in the control medium. Fourier transform infrared (FTIR) analysis confirmed the presence of a lipid profile functional group similar to biodiesel and major fatty acid methyl ester (FAME) profiling viz., tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), octadecanoic acid (C18:0), (Z)-Octadec-9-enoic acid (C18:1), linoleic acid (C18:2), alpha-linolenic acid (C18:3), cis-11-eicosenoic acid (C20:5), 13-cis-docosenoic acid (C22:6) on gas chromatography and mass spectrometric (GC-MS) analysis. A high productivity of lipid, biomass and biodiesel from isolated freshwater microalgae was found.

High carbon (CO2) supply leads to elevated intracellular acetyl CoA levels and increased lipid accumulation in Chlorella vulgaris

Carbon dioxide, the sole carbon source in phototrophic cultivation of microalgae, is the limiting factor for photosynthesis due to its low concentration in the atmosphere (0.04% v/v). We postulate that exposure to increased CO2 concentrations can increase the steady-state specific intracellular levels of carbon metabolic intermediates, including, the specific intracellular (si) levels of the precursor for fatty acid synthesis (acetyl CoA (AcCoA)), which in turn, appears to improve lipid accumulation. The effects of higher CO2 concentrations on Chlorella vulgaris were studied. At 2.6% v/v CO2, a 6-fold increase in volumetric lipid production was achieved. A 5.4-fold increase in the specific intracellular neutral lipid level (si-NL) from 9.6 to 52.3mg triolein (TO)/g biomass was also obtained. Si-AcCoA was significantly elevated at 2.6% CO2, and showed a 41, 25 and 27% increase over the air-sparged controls in the lag, log and stationary phases, respectively. Further, we show a quantitative empirical relationship between si-AcCoA and si-NL, which provides a new outlook for the design of strategies to improve lipid accumulation. In addition, favorable biodiesel characteristics were also obtained at higher CO2. Increased si-AcCoA with increase in CO2 concentrations, a related increase in lipid levels, and a quantitative empirical relationship between them have not been reported in the literature thus far.

Особенности метаболизма клеток Chlamydomonas reinhardtii CC-124 дикого штамма [137c] в условиях миксотрофного и фототрофного культивирования

Особенности метаболизма клеток Chlamydomonas reinhardtii CC-124 дикого штамма [137c] в условиях миксотрофного и фототрофного культивирования

Automation of a Nile red staining assay enables high throughput quantification of microalgal lipid production.

BackgroundWithin the context of microalgal lipid production for biofuels and bulk chemical applications, specialized higher throughput devices for small scale parallelized cultivation are expected to boost the time efficiency of phototrophic bioprocess development. However, the increasing number of possible experiments is directly coupled to the demand for lipid quantification protocols that enable reliably measuring large sets of samples within short time and that can deal with the reduced sample volume typically generated at screening scale. To meet these demands, a dye based assay was established using a liquid handling robot to provide reproducible high throughput quantification of lipids with minimized hands-on-time.ResultsLipid production was monitored using the fluorescent dye Nile red with dimethyl sulfoxide as solvent facilitating dye permeation. The staining kinetics of cells at different concentrations and physiological states were investigated to successfully down-scale the assay to 96 well microtiter plates. Gravimetric calibration against a well-established extractive protocol enabled absolute quantification of intracellular lipids improving precision from ±8 to ±2 % on average. Implementation into an automated liquid handling platform allows for measuring up to 48 samples within 6.5 h, reducing hands-on-time to a third compared to manual operation. Moreover, it was shown that automation enhances accuracy and precision compared to manual preparation. It was revealed that established protocols relying on optical density or cell number for biomass adjustion prior to staining may suffer from errors due to significant changes of the cells’ optical and physiological properties during cultivation. Alternatively, the biovolume was used as a measure for biomass concentration so that errors from morphological changes can be excluded.ConclusionsThe newly established assay proved to be applicable for absolute quantification of algal lipids avoiding limitations of currently established protocols, namely biomass adjustment and limited throughput. Automation was shown to improve data reliability, as well as experimental throughput simultaneously minimizing the needed hands-on-time to a third. Thereby, the presented protocol meets the demands for the analysis of samples generated by the upcoming generation of devices for higher throughput phototrophic cultivation and thereby contributes to boosting the time efficiency for setting up algae lipid production processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0433-7) contains supplementary material, which is available to authorized users.

Advances for Opaque PBR Internally Illuminated for Fiber Optic for Microalgae Production

The production of microalgae in laboratory systems is restricted almost exclusively since 500 mL to 20 liters tanks of transparent materials such as glass and plastic, under fluorescent lamps on shelves. In this work we developed a laboratory system which produced up to 50 liters of micro- algae cultivation with comparable productivity to the traditional system in the laboratory, with the potential to increase productivity scale and lower energy consumption per produced volume. The system is built with opaque plastic tank, and illuminated by Plastic Optics Fiber (POF) and LED. The quality of the biomass grown in the culture system on LED is comparable to the tradi- tional cultivation, with scale-up without increasing the occupied area, only with increased height of the tank. The productivity of the tank on LED to the strain Scenedesmus sp. with a phototrophic cultivation reached productivity of around 20 mg•L–1•d–1 and continuing studies may increase further.

Design and parallelisation of a miniature photobioreactor platform for microalgal culture evaluation and optimisation

Miniature photobioreactors (mPBr) represent a potential platform technology for the high-throughput, phototrophic cultivation of microalgae. This work describes the development and characterisation of a novel orbitally shaken twin-well mPBr, and its scale-out to a 24-well microplate format, suitable for optimisation of microalgae culture conditions. Fluid hydrodynamics, oxygen mass transfer coefficient (kLa) and light intensity distribution in the mPBr were first investigated as a function of orbital shaking frequency. High speed video analysis of the shaken wells indicated rapid fluid flow and good mixing while measured kLa values varied between 20 and 80h−1. Light intensity variation across the scaled-out platform was in the range ±20μmolm−2s−1. The use of the mPBr platform was demonstrated for optimisation of conditions for the batch cultivation of Chlorella sorokiniana. Using a modified tris-base phosphate (TBP) medium, the highest biomass concentration and productivity achieved were 9.2gL−1 and 2.5±0.2gL−1d−1 respectively at 5% CO2 with a light intensity of 380μmolm−2s−1. In general, cell growth rate and yield increased with increasing shaking frequency (up to 300rpm) while culture conditions had limited impact on pigment production. Overall, these results demonstrate the application of the mPBr for rapid optimisation of phototrophic culture conditions and establishment of high cell density cultures.

Features of Cell Metabolism of Chlamydomonas reinhardtii CC-124 Wild Strain [137c] Under Mixotrophic and Phototrophic Cultivation

Features of Cell Metabolism of Chlamydomonas reinhardtii CC-124 Wild Strain [137c] Under Mixotrophic and Phototrophic Cultivation

Fed-batch strategy for enhancing cell growth and C-phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation

The C-phycocyanin generated in blue-green algae Arthrospira platensis is gaining commercial interest due to its nutrition and healthcare value. In this study, the light intensity and initial biomass concentration were manipulated to improve cell growth and C-phycocyanin production of A.platensis in batch cultivation. The results show that low light intensity and high initial biomass concentration led to increased C-phycocyanin accumulation. The best C-phycocyanin productivity occurred when light intensity and initial biomass concentration were 300μmol/m2/s and 0.24g/L, respectively. The fed-batch cultivation proved to be an effective strategy to further enhance C-phycocyanin production of A.platensis. The results indicate that C-phycocyanin accumulation not only requires nitrogen-sufficient condition, but also needs other nutrients. The highest C-phycocyanin content (16.1%), production (1034mg/L) and productivity (94.8mg/L/d) were obtained when using fed-batch strategy with 5mM medium feeding.

Combination of algae and yeast fermentation for an integrated process to produce single cell oils.

Economic and ecological reasons cause the industry to develop new innovative bio-based processes for the production of oil as renewable feedstock. Petroleum resources are expected to be depleted in the near future. Plant oils as sole substituent are highly criticized because of the competitive utilization of the agricultural area for food and energy feedstock production. Microbial lipids of oleaginous microorganisms are therefore a suitable alternative. To decrease production costs of microbial lipids and gain spatial independence from industrial sites of CO2 emission, a combination of heterotrophic and phototrophic cultivation with integrated CO2 recycling was investigated in this study. A feasibility study on a semi-pilot scale was conducted and showed that the cultivation of the oleaginous yeast Cryptococcus curvatus on a 1.2-L scale was sufficient to supply a culture of the oleaginous microalgae Phaeodactylum tricornutum in a 21-L bubble column reactor with CO2 while single cell oils were produced in both processes due to a nutrient limitation.

Manipulation of light wavelength at appropriate growth stage to enhance biomass productivity and fatty acid methyl ester yield using Chlorella vulgaris

LEDs light offer several advantages over the conventional lamps, thereby being considered as the optimal light sources for microalgal cultivation. In this study, various light-emitting diodes (LEDs) especially red and blue color with different light wavelengths were employed to explore the effects of light source on phototrophic cultivation of Chlorella vulgaris. Blue light illumination led to significantly increased cell size, whereas red light resulted in small-sized cell with active divisions. Based on the discovery of the effect of light wavelengths on microalgal biology, we then applied appropriate wavelength at different growth stages; blue light was illuminated first and then shifted to red light. By doing so, biomass and lipid productivity of C. vulgaris could be significantly increased, compared to that in the control. These results will shed light on a novel approach using LED light for microalgal biotechnology.

Strategies to Improve Oil/Lipid Production of Microalgae in Outdoor Cultivation Using Vertical Tubular-type Photobioreactors

The indoor laboratory-scale microalgae culture was elevated to 50 liter in outdoor photobioreactors for the cultivation of an oil-rich microalga, Chlorella vulgaris ESP-31. Under phototrophic and photoheterotrophic conditions using CO2 and acetic acid as carbon source, respectively, the lipid productivity and lipid content obtained were 30-31mg/L/d and 35-44%, respectively. The effect of inoculum sizes on the performance of biomass and lipid production under phototrophic cultivation was also investigated. The lipid productivity of C. vulgaris ESP-31 was further improved to 47.6mg/L/d when a higher inoculum size of 0.70g/L was used. Finally, to avoid severe contamination problem during outdoor cultivation in the summer time, a two-stage lipid accumulation strategy was employed. The results show that the proposed two-stage process was able to avoid the bacterial contamination and enhanced the lipid content and lipid productivity of Chlorella vulgaris ESP-31.

Production of biodiesel from microalgae

In recent years, more attention has been paid to the use of third generation feedstocs for the production of biodiesel. One of the most promising sources of oil for biodiesel production are microalgae. They are unicellular or colonial photosynthetic organisms, with permanently increasing industrial application in the production of not only chemicals and nutritional supplements but also biodiesel. Biodiesel productivity per hectare of cultivation area can be up to 100 times higher for microalgae than for oil crops. Also, microalgae can grow in a variety of environments that are often unsuitable for agricultural purposes. Microalgae oil content varies in different species and can reach up to 77% of dry biomass, while the oil productivity by the phototrophic cultivation of microalgae is up to 122 mg/l/d. Variations of the growth conditions and the implementation of the genetic engineering can induce the changes in the composition and productivity of microalgal oil. Biodiesel from microalgae can be produced in two ways: by transesterification of oil extracted from biomass or by direct transesterification of algal biomass (so called in situ transesterification). This paper reviews the curent status of microalgae used for the production of biodiesel including their isolation, cultivation, harvesting and conversion to biodiesel. Because of high oil productivity, microalgae will play a significant role in future biodiesel production. The advantages of using microalgae as a source for biofuel production are increased efficiency and reduced cost of production. Also, microalgae do not require a lot of space for growing and do not have a negative impact on the global food and water supplies. Disadvantages of using microalgae are more difficult separation of biomass and the need for further research to develop standardized methods for microalgae cultivation and biodiesel production. Currently, microalgae are not yet sustainable option for the commercial production of biodiesel. First of all, the price of biodiesel from microalgae is still higher than the price of diesel due to high production costs.

Deceleration‐stats save much time during phototrophic culture optimization

In case of phototrophic cultures, photobioreactor costs contribute significantly to the total operating costs. Therefore one of the most important parameters to be determined is the maximum biomass production rate, if biomass or a biomass associated product is the desired product. This is traditionally determined in time consuming series of chemostat cultivations. The goal of this work is to assess the experimental time that can be saved by applying the deceleration stat (D-stat) technique to assess the maximum biomass production rate of a phototrophic cultivation system, instead of a series of chemostat cultures. A mathematical model developed by Geider and co-workers was adapted in order to describe the rate of photosynthesis as a function of the local light intensity. This is essential for the accurate description of biomass productivity in phototrophic cultures. The presented simulations demonstrate that D-stat experiments executed in the absence of pseudo steady-state (i.e., the arbitrary situation that the observed specific growth rate deviates <5% from the dilution rate) can still be used to accurately determine the maximum biomass productivity of the system. Moreover, this approach saves up to 94% of the time required to perform a series of chemostat experiments that has the same accuracy. In case more information on the properties of the system is required, the reduction in experimental time is reduced but still significant.

Phototrophic cultivation of a thermo-tolerant Desmodesmus sp. for lutein production: Effects of nitrate concentration, light intensity and fed-batch operation

Four indigenous thermo-tolerant Desmodesmus sp. strains were examined for their ability to produce lutein. Among them, Desmodesmus sp. F51 was the best strain for this purpose. The medium composition, nitrate concentration and light intensity were manipulated to improve the phototrophic growth and lutein production of Desmodesmus sp. F51. It was found that a nitrogen-sufficient condition was required for lutein accumulation, while a high light intensity enhanced cell growth but caused a decrease in the lutein content. The best cell growth and lutein production occurred when the light intensity and initial nitrate concentration were 600 μmol/m(2)/s and 8.8 mM, respectively. The fed-batch cultivation strategy was shown to further improve lutein production. The highest lutein productivity (3.56±0.10 mg/L/d) and content (5.05±0.20 mg/g) were obtained when pulse-feeding of 2.2 mM nitrate was employed. This study demonstrated the potential of using Desmodesmus sp. F51 as a lutein producer in practical applications.