The Impact of Calcium Depletion on Proliferation of Chlorella sorokiniana Strain DSCG150.

This study analyzed the effects of Ca2+ metal ions among culture medium components on the Chlorella sorokiniana strain DSCG150 strain cell growth. The C. sorokiniana strain DSCG150 strain grew based on a multiple fission cell cycle and growth became stagnant in the absence of metal ions in the medium, particularly Ca2+. Flow cytometry and confocal microscopic image analysis results showed that in the absence of Ca2+, cell growth became stagnant as the cells accumulated into four autospores and could not transform into daughter cells. Genetic analysis showed that the absence of Ca2+ caused upregulation of calmodulin (calA) and cell division control protein 2 (CDC2_1) genes, and downregulation of origin of replication complex subunit 6 (ORC6) and dual specificity protein phosphatase CDC14A (CDC14A) genes. Analysis of gene expression patterns by qRT-PCR showed that the absence of Ca2+ did not affect cell cycle progression up to 4n autospore, but it inhibited Chlorella cell fission (liberation of autospores). The addition of Ca2+ to cells cultivated in the absence of Ca2+ resulted in an increase in n cell population, leading to the resumption of C. sorokiniana growth. These findings suggest that Ca2+ plays a crucial role in the fission process in Chlorella.

High-Throughput In Vitro Screening of Changed Algal Community Structure Using the PhotoBiobox.

In a previous study, the sequential optimization and regulation of environmental parameters using the PhotoBiobox were demonstrated with high-throughput screening tests. In this study, we estimated changes in the biovolume-based composition of a polyculture built in vitro and composed of three algal strains: Chlorella sp., Scenedesmus sp., and Parachlorella sp. We performed this work using the PhotoBiobox under different temperatures (10-36°C) and light intensities (50-700 µmol/m-2/s-1) in air and in 5% CO2. In 5% CO2, Chlorella sp. exhibited better adaptation to high temperatures than in air conditions. Pearson's correlation analysis showed that the composition of Parachlorella sp. was highly related to temperature whereas Chlorella sp. and Scenedesmus sp. showed negative correlations in both air and 5% CO2. Furthermore, light intensity slightly affected the composition of Scenedesmus sp., whereas no significant effect was observed in other species. Based on these results, it is speculated that temperature is an important factor in influencing changes in algal polyculture community structure (PCS). These results further confirm that the PhotoBiobox is a convenient and available tool for performance of lab-scale experiments on PCS changes. The application of the PhotoBiobox in PCS studies will provide new insight into polyculture-based ecology.

Genome-wide high-throughput screening of interactive bacterial metabolite in the algal population using Escherichia coli K-12 Keio collection.

Algae-bacteria interaction is one of the main factors underlying the formation of harmful algal blooms (HABs). The aim of this study was to develop a genome-wide high-throughput screening method to identify HAB-influenced specific interactive bacterial metabolites using a comprehensive collection of gene-disrupted E. coli K-12 mutants (Keio collection). The screening revealed that a total of 80 gene knockout mutants in E. coli K-12 resulted in an approximately 1.5-fold increase in algal growth relative to that in wild-type E. coli. Five bacterial genes (lpxL, lpxM, kdsC, kdsD, gmhB) involved in the lipopolysaccharide (LPS) (or lipooligosaccharide, LOS) biosynthesis were identified from the screen. Relatively lower levels of LPS were detected in these bacteria compared to that in the wild-type. Moreover, the concentration-dependent decrease in microalgal growth after synthetic LPS supplementation indicated that LPS inhibits algal growth. LPS supplementation increased the 2,7-dichlorodihydrofluorescein diacetate fluorescence, as well as the levels of lipid peroxidation-mediated malondialdehyde formation, in a concentration-dependent manner, indicating that oxidative stress can result from LPS supplementation. Furthermore, supplementation with LPS also remarkably reduced the growth of diverse bloom-forming dinoflagellates and green algae. Our findings indicate that the Keio collection-based high-throughput in vitro screening is an effective approach for the identification of interactive bacterial metabolites and related genes.

Two-stage cultivation strategy for the improvement of pigment productivity from high-density heterotrophic algal cultures.

Herein, a two-stage cultivation process was devised to overcome low pigment content of algal biomass grown in heterotrophy. Post-treatment conditions (i.e., light intensity, temperature, pH and salinity) were initially tested for dense heterotrophically-grown Chlorella sp. HS2 cultures in a multi-channel photobioreactor (mcPBR), and the results clearly indicated the influence of each abiotic factor on algal pigment production. Subsequently, the optimal post-treatment conditions (i.e., 455 µmol m-2 s-1, 34.8℃, pH 8.23 and 0.7% (w/v) salinity), in which highest accumulation of algal pigments is expected, were identified using Response Surface Methodology (RSM). Compared to the control cultures grown in mixotrophy for the same duration of entire two-stage process, the results indicated a significantly higher pigment productivity (i.e., 167.5 mg L-1 day-1) in a 5-L fermenter following the post-treatment at optimal conditions. Collectively, these results suggest that the post-treatment of heterotrophic cultures can be successfully deployed to harness the nascent algae-based bioeconomy.

Enhancement of ß-carotene production by regulating the autophagy-carotenoid biosynthesis seesaw in Chlamydomonas reinhardtii.

This work aimed to demonstrate a new strategy for enhancing the production of carotenoids through the regulation of seesaw cross-talk between autophagy and carotenoid biosynthesis pathways in Chlamydomonas reinhardtii. Autophagy-related ATG1 and ATG8 genes were first silenced using artificial microRNA, which in turn reduced the mRNA expression of ATG1 and ATG8 by 84.4% and 74.3%, respectively. While ATG1 kinase controls early step in autophagy induction and ATG8 is an essential factor for the downstream formation of autophagosome membranes, the decreased expression of these genes led to a 2.34-fold increase in the amount of ß-carotene content (i.e., 23.75 mg/g DCW). Furthermore, all mutants seemed to exhibit greater biodiesel properties than that of wild-type due to increased accumulation of saturated and monounsaturated fatty acids. These results support the role of autophagy in regulating the production of valuable metabolites, which could contribute to uplifting the economic outlook of nascent algal biorefinery.

Impairment of starch biosynthesis results in elevated oxidative stress and autophagy activity in Chlamydomonas reinhardtii.

Autophagy is a self-degradation system wherein cellular materials are recycled. Although autophagy has been extensively studied in yeast and mammalian systems, integrated stress responses in microalgae remain poorly understood. Accordingly, we carried out a comparative study on the oxidative stress responses of Chlamydomonas reinhardtii wild-type and a starchless (sta6) mutant previously shown to accumulate high lipid content under adverse conditions. To our surprise, the sta6 mutant exhibited significantly higher levels of lipid peroxidation in the same growth conditions compared to controls. The sta6 mutant was more sensitive to oxidative stress induced by H2O2, whereas the wild-type was relatively more resistant. In addition, significantly up-regulated autophagy-related factors including ATG1, ATG101, and ATG8 were maintained in the sta6 mutant regardless of nitrogen availability. Also, the sta6 mutant exhibited relatively higher ATG8 protein level compared to wild-type under non-stress condition, and quickly reached a saturation point of autophagy when H2O2 was applied. Our results indicate that, in addition to the impact of carbon allocation, the increased lipid phenotype of the sta6 mutant may result from alterations in the cellular oxidative state, which in turn activates autophagy to clean up oxidatively damaged components and fuel lipid production.

Nitrogen modulation under chemostat cultivation mode induces biomass and lipid production by Chlorella vulgaris and reduces antenna pigment accumulation.

Algal growth limitation in large-scale cultivation mostly results from high level synthesis of photosynthetic pigments, owing to self-shading effects and attenuation of light distribution. To overcome this problem, here we investigated the influence of nitrogen modulation on changes in antenna pigments as well as biomass and lipid production by Chlorella vulgaris under a chemostat continuous cultivation mode. The production of algal antenna pigments, including chlorophylls and carotenoids, was promoted in a total nitrogen (TN) concentration-dependent manner. Maximum algal biomass and lipid production were obtained from 70 mg/L of TN concentration along with a significant increase in light transmittance and reduction in antenna pigments. Furthermore, the composition of polyunsaturated fatty acids remarkably augmented at low TN concentrations. These results suggest that the reduction in algal antenna pigment synthesis via modulation of nitrogen concentration may serve as an effective strategy to enhance algal biomass and lipid production.