Large-scale Outdoor Cultivation Research Articles

Optimizing cultivation strategies and scaling up for fucoxanthin production using Pavlova sp.

The microalgal-based production of fucoxanthin has emerged as an imperative research endeavor due to its antioxidant, and anticancer properties. In this study, three brown marine microalgae, namely Skeletonema costatum, Chaetoceros gracilis, and Pavlova sp., were screened for fucoxanthin production. All strains displayed promising results, with Pavlova sp. exhibiting the highest fucoxanthin content (27.91 mg/g) and productivity (1.16 mg/L·day). Moreover, the influence of various cultivation parameters, such as culture media, salinity, sodium nitrate concentration, inoculum size, light intensity, and iron concentration, were investigated and optimized, resulting in a maximum fucoxanthin productivity of 7.89 mg/L·day. The investigation was further expanded to large-scale outdoor cultivation using 50 L tubular photobioreactors, illustrating the potential of Pavlova sp. and the cultivation process for future commercialization. The biomass and fucoxanthin productivity for the large-scale cultivation were 70.7 mg/L·day and 4.78 mg/L·day, respectively. Overall, the findings demonstrated considerable opportunities for fucoxanthin synthesis via microalgae cultivation and processing.

Microalgae biofuels: illuminating the path to a sustainable future amidst challenges and opportunities.

The development of microalgal biofuels is of significant importance in advancing the energy transition, alleviating food pressure, preserving the natural environment, and addressing climate change. Numerous countries and regions across the globe have conducted extensive research and strategic planning on microalgal bioenergy, investing significant funds and manpower into this field. However, the microalgae biofuel industry has faced a downturn due to the constraints of high costs. In the past decade, with the development of new strains, technologies, and equipment, the feasibility of large-scale production of microalgae biofuel should be re-evaluated. Here, we have gathered research results from the past decade regarding microalgae biofuel production, providing insights into the opportunities and challenges faced by this industry from the perspectives of microalgae selection, modification, and cultivation. In this review, we suggest that highly adaptable microalgae are the preferred choice for large-scale biofuel production, especially strains that can utilize high concentrations of inorganic carbon sources and possess stress resistance. The use of omics technologies and genetic editing has greatly enhanced lipid accumulation in microalgae. However, the associated risks have constrained the feasibility of large-scale outdoor cultivation. Therefore, the relatively controllable cultivation method of photobioreactors (PBRs) has made it the mainstream approach for microalgae biofuel production. Moreover, adjusting the performance and parameters of PBRs can also enhance lipid accumulation in microalgae. In the future, given the relentless escalation in demand for sustainable energy sources, microalgae biofuels should be deemed a pivotal constituent of national energy planning, particularly in the case of China. The advancement of synthetic biology helps reduce the risks associated with genetically modified (GM) microalgae and enhances the economic viability of their biofuel production.

Multifaceted strategies for economic production of microalgae Haematococcus pluvialis-derived astaxanthin via direct conversion of CO2

Owing to its strong antioxidant properties, astaxanthin has a high market price in the nutraceutical and pharmaceutical fields, and its demand is increasing. Furthermore, with an increase in the demand for green technology, astaxanthin production through direct CO2 conversion using the autotrophic green microalga Haematococcus pluvialis as a bio-platform has received much attention. Large-scale outdoor cultivation of H. pluvialis using waste CO2 sources and sunlight can secure sustainability and improve economic efficiency. However, low strain performance, reduced light utilization because of increased cell density, and inefficient transfer of gaseous CO2 into liquid culture broth hinder its large-scale commercialization of astaxanthin. Herein, we presented a multifaceted strategy, including the development of high-efficiency strains, a culture system for astaxanthin accumulation, and astaxanthin extraction from biomass, for economically producing astaxanthin from H. pluvialis through direct CO2 conversion. Future perspectives were presented by comparing and analyzing various previous studies conducted using the latest technology.

Microbiota associated with the large-scale outdoor cultivation of the cyanobacterium Synechococcus sp. PCC 7002

The model euryhaline cyanobacterium Synechococcus sp. PCC 7002 has potential as a platform for biotechnological applications because of its genetic amenability and fast growth rates. The purpose of this work was to measure the outdoor productivity metrics of Synechococcus sp. PCC 7002, which we tested by cultivation in 110 L flat-panel photobioreactors and 1025 L open raceway ponds at the Algae Testbed Public Private Partnership (ATP3) algae culturing testbed site at the Arizona Center for Algae Technology and Innovation (AzCATI). This work was carried out during the summer growing season when ambient temperatures and light availability are highest to support maximum photoautotrophic biomass yields. Modifications to the traditional A+ laboratory growth media for Synechococcus sp. PCC 7002 included the use of urea as a nitrogen source, and CO2 sparging for automated pH control in open ponds. Synechococcus showed robust growth in flat-panel photobioreactors, which are suited for the containment of modified organisms; however, cultivation in the open-raceway pond system was challenging due to pond crashes. We investigated the microbiota associated with Synechococcus pond cultures using Illumina amplicon sequencing of the 16S rRNA genes of bacteria and archaea and the 18S rRNA genes of microbial eukaryotes, to identify potential competitor and predator genera. These results pointed to likely predation of Synechococcus by a ciliated protozoan of the genus Euplotes. Development of crop protection strategies will be crucial for enabling the mass cultivation of Synechococcus in open pond systems.

Biocontainment of Engineered Synechococcus elongatus PCC 7942 for Photosynthetic Production of α-Farnesene from CO2.

Biocontainment systems have been developed to mitigate the concerns regarding biosafety and environmental risk because of the possible escape of genetically modified organisms into the environment following large-scale outdoor cultivation. Here, we present a biocontainment system entailing genetically modified Synechococcus elongatus PCC 7942, also engineered for α-farnesene production using a de-evolutionary strategy. In this approach, the gene cluster encoding the β-carboxysome and the associated carbon concentrating mechanism (CCM) were deleted in the α-farnesene-producing cyanobacteria, resulting in no cell growth and no α-farnesene production at ambient CO2 concentrations (100% air bubbling). However, cell growth and α-farnesene production were detected in the CCM-deficient strains at high CO2 concentrations (5% CO2 [v/v], 10% CO2 [v/v]), albeit at levels lower than those of the parental control. To overcome this limitation, the overexpression of carbonic anhydrase and bicarbonate transporter genes in the CCM-deficient strains restored cell growth and the production level of α-farnesene (5.0 ± 0.6 mg/L) to that of the parental control. The production of α-farnesene in the later strains strictly depended on CO2 concentration in the photobioreactor and did not rely on a chemical induction process. Thus, next generation bio-solar cell factories could be promoted with the suggested biocontainment system.

Deciphering and engineering high-light tolerant cyanobacteria for efficient photosynthetic cell factories

Development and utilization of “liquid sunshine” could be one of key solutions to deal with the issues of fossil fuel depletion and increasing carbon dioxide. Cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis, and their activity accounts for ~25% of the total carbon fixation on earth. More importantly, besides their traditional roles as primary producers, cyanobacteria could be modified as “photosynthetic cell factories” to produce renewable fuels and chemicals directly from CO 2 driven by solar energy, with the aid of cutting-edging synthetic biology technology. Towards their large-scale biotechnological application in the future, many challenges still need to be properly addressed, among which is cyanobacterial cell factories inevitably suffer from high light (HL) stress during large-scale outdoor cultivation, resulting in photodamage and even cell death, limiting their productivity. In this review, we critically summarized recent progress on deciphering molecular mechanisms to HL and developing HL-tolerant chassis in cyanobacteria, aiming at facilitating construction of HL-resistant chassis and promote the future application of the large-scale outdoor cultivation of cyanobacterial cell factories. Finally, the future directions on cyanobacterial chassis engineering were discussed.

A strategy for stimulating astaxanthin and lipid production in Haematococcus pluvialis by exogenous glycerol application under low light

In large-scale outdoor cultivation, Haematococcus pluvialis cultures are frequently exposed to low light caused by continuous rain conditions. Low light significantly restricts biomass and astaxanthin accumulation, thereby decreasing astaxanthin productivity. In this study, the effects of exogenous glycerol on the growth, photosynthesis, and astaxanthin and lipid accumulation in H. pluvialis under low light were investigated. A strategy was developed for promoting astaxanthin and lipid production in H. pluvialis under low light by the application of exogenous glycerol. Under low light, exogenous glycerol did not change the biomass and photosynthetic capacity of H. pluvialis. The addition of 1 mL L−1 glycerol increased total lipid content by 36.8% by increasing the formation of fatty acids during an 11-day growth period. Furthermore, exogenous glycerol could directly accelerate astaxanthin production under low light by increasing the formation of substrates (e.g. pyruvate). Meanwhile, the elevated fatty acid level due to the addition of glycerol facilitated the esterification of astaxanthin, thereby accelerating the biosynthesis of astaxanthin indirectly, which is indicated by the fact that exogenous glycerol enhanced astaxanthin esters content significantly under low light. All these results suggest that H. pluvialis has the ability to utilize glycerol for astaxanthin and lipid production under low light.

Transcriptomic insights into the heat stress response of Dunaliella bardawil

The halophilic green alga Dunaliella bardawil has been used for commercial production of natural β-carotene by large-scale outdoor cultivation, which often suffers from heat stress especially at noon in hot summers. In this study, the effects of heat stress on cell growth, pigment contents, and activities of antioxidant system in D. bardawil were studied, and RNA-seq experiment was conducted to analyze the transcriptional response to heat stress (42 °C for 2 h) in D. bardawil. High temperature (42 °C) for short time treatment (≤3 h) did not severely affect the cell growth and pigment accumulation of D. bardawil. Multiple genes encoding heat shock proteins for protein folding and antioxidant enzymes against toxic reactive oxygen species were substantially up-regulated significantly under heat stress. D. bardawil cells tended to shift from aerobic to glycolytic metabolism for energy production to increase survival chances under heat stress. Furthermore, the enrichment of ascorbate-glutathione cycle, up-regulation of genes responsible for chloroplast membranes, and changes in lipid characteristics like carbon chain length and unsaturation degree could play a vital role in achieving thermotolerance of D. bardawil. Taken together, this study improved our understanding of the molecular mechanisms of heat stress responses in D. bardawil.

Biomass and lipid production of a novel freshwater thermo-tolerant mutant strain of Chlorella pyrenoidosa NCIM 2738 in seawater salinity recycled medium

Micro-algae have significant potential for the production of biofuel. It has been previously shown that existing processes of producing micro-algal biofuels are not yet economically viable at commercial scale. Fundamentally, this process will require the implementation of inexpensive harvesting methods and productive algal strains that are tolerant to harsh conditions. The use of freshwater in the large-scale cultivation of microalgae is currently unsustainable; hence, the studies which target the use of seawater medium can reduce the demand of freshwater. Chlorella pyrenoidosa has been extensively studied in freshwater. However, information on the cultivation of C. pyrenoidosa for growth productivity in recycled seawater medium, generated after harvesting through autoflocculation is scarce. In this context, the present study investigates the effect of nutrients sufficient seawater medium following reuse of nutrients exhausted seawater medium on the biochemical composition, biomass and lipid productivities of an in-house developed, thermotolerant strain of C. pyrenoidosa under high temperature (45 °C), outdoor culture conditions. The strain C. pyrenoidosa M18 was found to thrive in seawater medium resulting in increased biomass and lipid productivity. Lipid productivity in seawater medium exceeded 51 mg/Ld−1 which was nearly 2-fold greater than freshwater. Furthermore, the strain was able to grow well and autoflocculate at recycled seawater medium giving biomass and lipid productivities, respectively of 338 mgL−1d−1 and 66 mgL−1d−1. The dry cell weight comprised of 28–32% carbohydrate content when cultivated in recycled seawater medium. Additionally, exposure to high salinity led to an enhanced chlorophyll content as well as increased Fv/Fm ratio. The auto-flocculating property of thermotolerant C. pyrenoidosa M18 strain, in addition to the high biomass and lipid productivities in nutrient exhausted recycled seawater medium, make this microalga an excellent candidate for large-scale outdoor cultivation for biodiesel production.

Modeling Microalgae Productivity in Industrial-Scale Vertical Flat Panel Photobioreactors.

Potentially achievable biomass yields are a decisive performance indicator for the economic viability of mass cultivation of microalgae. In this study, a computer model has been developed and applied to estimate the productivity of microalgae for large-scale outdoor cultivation in vertical flat panel photobioreactors. Algae growth is determined based on simulations of the reactor temperature and light distribution. Site-specific weather and irradiation data are used for annual yield estimations in six climate zones. Shading and reflections between opposing panels and between panels and the ground are dynamically computed based on the reactor geometry and the position of the sun. The results indicate that thin panels (≤0.05 m) are best suited for the assumed cell density of 2 g L-1 and that reactor panels should face in north-south direction. Panel spacings of 0.4-0.75 m at a panel height of 1 m appear most suitable for commercial applications. Under these preconditions, yields of around 10 kg m-2 a-1 are possible for most locations in the U.S. Only in hot climates significantly lower yields have to be expected, as extreme reactor temperatures limit overall productivity.

Chronic alcohol consumption alters transcriptional profiles and mucosal cytokine production of intestinal lamina propria lymphocytes

Micro-algae have significant potential for the production of biofuel. It has been previously shown that existing processes of producing micro-algal biofuels are not yet economically viable at commercial scale. Fundamentally, this process will require the implementation of inexpensive harvesting methods and productive algal strains that are tolerant to harsh conditions. The use of freshwater in the large-scale cultivation of microalgae is currently unsustainable; hence, the studies which target the use of seawater medium can reduce the demand of freshwater. Chlorella pyrenoidosa has been extensively studied in freshwater. However, information on the cultivation of C. pyrenoidosa for growth productivity in recycled seawater medium, generated after harvesting through autoflocculation is scarce. In this context, the present study investigates the effect of nutrients sufficient seawater medium following reuse of nutrients exhausted seawater medium on the biochemical composition, biomass and lipid productivities of an in-house developed, thermotolerant strain of C. pyrenoidosa under high temperature (45 °C), outdoor culture conditions. The strain C. pyrenoidosa M18 was found to thrive in seawater medium resulting in increased biomass and lipid productivity. Lipid productivity in seawater medium exceeded 51 mg/Ld−1 which was nearly 2-fold greater than freshwater. Furthermore, the strain was able to grow well and autoflocculate at recycled seawater medium giving biomass and lipid productivities, respectively of 338 mgL−1d−1 and 66 mgL−1d−1. The dry cell weight comprised of 28–32% carbohydrate content when cultivated in recycled seawater medium. Additionally, exposure to high salinity led to an enhanced chlorophyll content as well as increased Fv/Fm ratio. The auto-flocculating property of thermotolerant C. pyrenoidosa M18 strain, in addition to the high biomass and lipid productivities in nutrient exhausted recycled seawater medium, make this microalga an excellent candidate for large-scale outdoor cultivation for biodiesel production.

Evaluation of phenotype stability and ecological risk of a genetically engineered alga in open pond production

Genetically engineered (GE) algae offer the promise of producing food, fuel, and other valuable products with reduced requirements for land and fresh water. While the gains in productivity measured in GE terrestrial crops are predicted to be mirrored in GE algae, the stability of phenotypes and ecological risks posed by GE algae in large-scale outdoor cultivation remain unknown. Here, we describe the first US Environmental Protection Agency (EPA)-sanctioned experiment aimed at understanding how GE algae perform in outdoor cultivation. Acutodesmus dimorphus was genetically engineered by the addition of two genes, one for enhanced fatty acid biosynthesis, and one for recombinant green fluorescence protein (GFP) expression; both the genes and their associated phenotypes were maintained during fifty days of outdoor cultivation. We also observed that while the GE algae dispersed from the cultivation ponds, colonization of the trap ponds by the GE strain declined rapidly with increasing distance from the source cultivation ponds. In contrast, many species of indigenous algae were found in every trap pond within a few days of starting the experiment. When inoculated in water from five local lakes, the GE algae's effect on biodiversity, species composition, and biomass of native algae was indiscernible from those of the wild-type (wt) progenitor algae, and neither the GE nor wt algae were able to outcompete native strains. We conclude that GE algae can be successfully cultivated outdoors while maintaining GE traits, and that for the specific GE algal strain tested here they did not outcompete or adversely impact native algae populations when grown in water taken from local lakes. This study provides an initial evaluation of GE algae in outdoor cultivation and a framework to evaluate GE algae risks associated with outdoor GE algae production.

Truncated light-harvesting chlorophyll antenna size in Chlorella vulgaris improves biomass productivity

Microalgae have been proposed as eco-friendly feedstocks for biodiesel production, because they accumulate large amounts of lipids and increase their biomass through photosynthesis. However, the photosynthetic efficiency of microalgae is too low for this strategy to be economically feasible. In an effort to overcome this issue, random mutants with reduced chlorophyll antenna size were generated by ethyl methanesulfonate (EMS)-mediated mutagenesis of Chlorella vulgaris. The antenna size mutant, herein designated E5, exhibited 56.5 and 75.8 % decreases in chlorophyll a and b contents, respectively, with significant reductions in the expression levels of peripheral light-harvesting antenna proteins in photosystem II. The saturated photosynthetic activity and electron transport rate of the E5 mutant were significantly higher and also showed reduced non-photochemical quenching (NPQ), compared to those of the wild type. Consequentially, the E5 mutant cultures achieved 44.5 % improvement in biomass productivity under high light (200 μmol photons m−2 s−1). These results suggest that improving the photosynthetic efficiency of microalgae could greatly enhance their biomass production, and such mutant strains can be applicable for large-scale outdoor cultivation which is typically exposed to high light intensity.

Utilization of gaseous emissions from a methanol plant for cultivation of Acutodesmus obliquus

This study aimed to culture the green alga Acutodesmus obliquus utilizing the gaseous emissions containing a high concentration of CO2 (99.13 %) from a methanol plant and study the tolerance of microalgae. The effect of CO2 concentration, aeration rate, inoculum concentration, intermittent sparging, and nitrogen sources on the growth of A. obliquus was examined. Acutodesmus obliquus also was cultivated in a 500-L pilot outdoor tubular photobioreactor (OTP) to advance the laboratory scale system to outdoor scale-up applications. The results showed that A. obliquus could tolerate high CO2 concentrations of 50 %, and a maximum biomass of 0.935 g L−1 (dry weight) was achieved at 20 % CO2. An aeration rate of 500 mL min−1, inoculum concentration (optical density at 680 nm [OD680] = 0.3), and intermittent sparging of 10 min per 2 h enhanced growth to the optimum and influenced culture pH and photosynthesis. Urea as a nitrogen source was shown to be more beneficial to cell growth. A urea concentration of 0.3 g L−1 and an N/P ratio of 15 led to maximum biomass accumulation thus enhancing the gaseous emission utilization efficiency. In conclusion, this work demonstrated that gaseous emissions containing high concentration of CO2 from a methanol plant could be directly introduced into A. obliquus cultures and that A. obliquus was suitable well for large-scale outdoor cultivation in a tubular photobiorecator.

Isolation and screening of euryhaline Tetraselmis spp. suitable for large-scale outdoor culture in hypersaline media for biofuels

Natural saline lakes in Western Australia were sampled for microalgae species and strains with potential for large-scale outdoor cultivation over a wide range of salinities for biofuels production. Using a rational isolation and screening process, several Tetraselmis strains (Chlorophyta, Chlorodendrales) with a broad range of salinity tolerance were identified and were characterised further for their potential for biofuels production. Specific growth rates increased from 0.8 to 1.2 days−1 when the medium salinity was decreased from 11 to 3 % (w/v) NaCl (1.88 to 0.51 M NaCl) in batch cultivation mode, thereby indicating quick adaptation to large salinity changes. In general, ash-free dry weight (AFDW), total lipid, protein and carbohydrate contents per cell were highest in the early stages of growth. Salinity increases led to an increase in cell AFDW, with the highest mean maximum of 2555 ± 659 pg AFDW.cell−1 at 11 % (w/v) NaCl in the strains Tetraselmis MUR 167 and MUR 219 which had been in culture for many years, as compared to the mean maximum of 981 ± 141 pg AFDW.cell−1 the in newly isolated strains MUR 230, 231, 232 and 233. Similar observations on total lipid, protein and carbohydrate content per cell were made between the two groups of strains. Overall, all strains yielded high biomass and total lipid productivities over a very wide range of salinities without large variation in their gross biochemical composition and growth pattern. Based on AFDW and total lipid productivity data, the order of preference for selecting strains for further investigation for large-scale culture was MUR 231 > MUR 233 > MUR 219 > MUR 230 > MUR 232 > MUR 167. The Tetraselmis spp. were also very competitive as shown by the outdoor cultivation of diatom, Halamphora coffeaeformis MUR 158, in parallel with Tetraselmis sp. MUR 167 which resulted in the diatom being outcompeted by the green alga. Our results demonstrate the high commercial potential of euryhaline Tetraselmis spp. for cultivation over a broad range of salinity in outdoor cultures.