Endophyte Bacillus tequilensis improves the growth of microalgae Haematococcus lacustris by regulating host cell metabolism.

This study identified an endosymbiotic bacterium, Bacillus tequilensis, residing within the cells of the microalga Haematococcus lacustris through 16S rRNA analysis. To confirm the optimal interactive conditions between H. lacustris and B. tequilensis, the effects of different ratios of cells using H. lacustris of different growth stages were examined. Under optimized conditions, the cell density, dry weight, chlorophyll content, and astaxanthin content of H. lacustris increased significantly, and the fatty acid content improved 1.99-fold. Microscopy demonstrated the presence of bacteria within the H. lacustris cells. The interaction upregulated amino acid and nucleotide metabolism in H. lacustris. Interestingly, muramic and phenylacetic acids were found exclusively in H. lacustris cells in the presence of B. tequilensis. Furthermore, B. tequilensis delayed pigment degradation in H. lacustris. This study reveals the impact of the endosymbiont B. tequilensis on the metabolism of H. lacustris and offers new perspectives on the symbiotic relationship between them.

Polyethylenimine linked with chitosan improves astaxanthin production in Haematococcus pluvialis.

Astaxanthin is receiving increasing interest as an antioxidant and high value-added secondary metabolite. Haematococcus pluvialis is the main source for astaxanthin production, and many studies are being conducted to increase the production of astaxanthin. In this study, we linked polyethylenimine (PEI) with chitosan to maintain astaxanthin-inducing ability while securing the recyclability of the inducer. Astaxanthin accumulation in H. pluvialis was induced to 86.4 pg cell-1 with the PEI-chitosan fiber (PCF) treatment prepared by cross-linking of 10 µM PEI and low molecular weight (MW) chitosan via epichlorohydrin. PEI concentration affected the astaxanthin accumulation, whereas the MW of chitosan did not. In addition, the PCF treatment in H. pluvialis increased the reactive oxygen species (ROS) content in cells, thereby upregulating the transcription of enzymes involved in astaxanthin biosynthesis. PCF can be reused multiple times with the maintenance of over 90% of the astaxanthin production efficiency. This study offers a reusable PCF stimulation strategy for enhancing natural astaxanthin content, and PCF treatment will easily increase the production scale or reduce production costs by using recyclability that is not available in current methods. KEY POINTS: • Polyethylenimine-chitosan fiber (PCF) was applied to Haematococcus pluvialis • PCF promotes astaxanthin accumulation by enhancing oxidative stress in H. pluvialis • PCF can be reused multiple times with maintaining over 90% production efficiency.

Establishment of ultrasonic stimulation to enhance growth of Haematococcus lacustris.

In this study, ultrasonication at a frequency of 40 kHz was used to shorten the sonication period and enhance the growth of Haematococcus lacustris. To confirm the optimal conditions, the effects of ultrasound output and treatment interval were examined. Under optimal conditions (20 W and 15-day cycle), the maximum cell density and chlorophyll content were 66.75 × 104 cells mL-1 and 36.54 mg g-1, respectively, which were increased by 50.00% and 39.01%, respectively, compared to the control. Transmission electron microscopy analysis showed that ultrasonication caused tiny cracks in the W4 and W6 strata but did not disrupt the inner W2 layer. Additionally, RT-qPCR analysis showed that ultrasonication upregulated both cell division and nitrogen uptake. No difference were detected in the composition or quantity of fatty acids. This study demonstrates a novel ultrasonic approach for enhancing the growth of H. lacustris.

Contributions of the microbiome to intestinal inflammation in a gut-on-a-chip.

The intestinal microbiome affects a number of biological functions of the organism. Although the animal model is a powerful tool to study the relationship between the host and microbe, a physiologically relevant in vitro human intestinal system has still unmet needs. Thus, the establishment of an in vitro living cell-based system of the intestine that can mimic the mechanical, structural, absorptive, transport and pathophysiological properties of the human intestinal environment along with its commensal bacterial strains can promote pharmaceutical development and potentially replace animal testing. In this paper, we present a microfluidic-based gut model which allows co-culture of human and microbial cells to mimic the gastrointestinal structure. The gut microenvironment is recreated by flowing fluid at a low rate (21 µL/h) over the microchannels. Under these conditions, we demonstrated the capability of gut-on-a-chip to recapitulate in vivo relevance epithelial cell differentiation including highly polarized epithelium, mucus secretion, and tight membrane integrity. Additionally, we observed that the co-culture of damaged epithelial layer with the probiotics resulted in a substantial responded recovery of barrier function without bacterial overgrowth in a gut-on-a-chip. Therefore, this gut-on-a-chip could promote explorations interaction with host between microbe and provide the insights into questions of fundamental research linking the intestinal microbiome to human health and disease.

Microalgal secondary metabolite productions as a component of biorefinery: A review.

The interest in developing microalgae for industrial use has been increasing because of concerns about the depletion of petroleum resources and securing sustainable energy sources. Microalgae have high biomass productivity and short culture periods. However, despite these advantages, various barriers need to be overcome for industrial applications. Microalgal cultivation has a high unit price, thus rendering industrial application difficult. It is indispensably necessary to co-produce their primary and secondary metabolites to compensate for these shortcomings. In this regard, this article reviews the following aspects, (1) co-production of primary and secondary metabolites in microalgae, (2) induction methods for the promotion of the biosynthesis of secondary metabolites, and (3) perspectives on the co-production and co-extraction of primary and secondary metabolites. This paper presents various approaches for producing useful metabolites from microalgae and suggests strategies that can be utilized for the co-production of primary and secondary metabolites.

Semi-continuous immobilized cultivation of Porphyridium cruentum for sulfated polysaccharides production.

In this study, semi-continuous immobilized cultivation of Porphyridium cruentum through calcium alginate beads was performed for sulfated polysaccharides (SPs) production. The cell biomass and daily SPs productivity in the calcium alginate bead immobilized culture were increased by up to 79 ± 3.4% and 45.6 ± 3.2%, compared to those in the control, respectively. Furthermore, simultaneous application of immobilization and blue wavelength illumination further increased the phycobiliproteins content by 260 ± 9%, compared to those in the control. Similarly, nutrient deficiencies in combination with immobilization increased daily SPs productivity by about twice that of the control. The chemical composition and biological activity of the extracellular polymeric substances produced through immobilization were similar to those of the control. This study suggests the potential application of calcium alginate beads-based immobilization for continuous and high-efficiency SPs production using P. cruentum.

A strategic approach to apply bacterial substances for increasing metabolite productions of Euglena gracilis in the bioreactor.

Bacterial extracellular polymeric substances (EPS) are promising materials that have a role in enhancing growth, metabolite production, and harvesting efficiency. However, the validity of the EPS effectiveness in scale-up cultivation of microalgae is still unknown. Therefore, in order to verify whether the bacterial metabolites work in the scale-up fermentation of microalgae, we conducted a bioreactor fermentation following the addition of bacterial EPS derived from the marine bacterium, Pseudoalteromonas sp., to Euglena gracilis. Various culture strategies (i.e., batch, glucose fed-batch, and glucose and EPS fed-batch) were conducted to maximize metabolite production of E. gracilis in scale-up cultivation. Consequently, biomass and paramylon concentrations in the continuous glucose and EPS-treated culture were enhanced by 3.0-fold and 4.2-fold (36.1 ± 1.4 g L-1 and 25.6 ± 0.1 g L-1), respectively, compared to the non-treated control (12.0 ± 0.3 g L-1 and 6.1 ± 0.1 g L-1). Also, the supplementation led to the enhanced concentrations of α-tocopherols and total fatty acids by 3.7-fold and 2.8-fold, respectively. The harvesting efficiency was enhanced in EPS-supplemented cultivation due to the flocculation of E. gracilis. To the best of our knowledge, this is the first study that verifies the effect of bacterial EPS in scale-up cultivation of microalgae. Also, our results showed the highest paramylon productivity than any other previous reports. The results obtained in this study showed that the scale-up cultivation of E. gracilis using bacterial EPS has the potential to be used as a platform to guide further increases in scale and in the industrial environment. KEY POINTS: Effect of EPS on Euglena gracilis fermentation was tested in bioreactor scale. EPS supplement was effective for the paramylon, α-tocopherol, and lipid production. EPS supplement induced the flocculation of E. gracilis.

Enhancement of phycoerythrin productivity in Porphyridium purpureum using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 ribonucleoprotein system.

Genetic manipulation of the Porphyridium sp. may increase the production of phycoerythrin. Since phycobiliproteins capture and transfer energy to both photosystems (PS I and PS II), it was hypothesized that the gene mutation involved increases phycoerythrin synthesis. The gene encoding chlorophyll synthase (CHS1) was selected as chlorophyll synthase plays an important role in photosynthesis, mediating the final process of chlorophyll synthesis. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 ribonucleoprotein (CRISPR/Cas9 RNP) delivery system was used to generate the chlorophyll synthase loss-of-function mutants (Δchs1). Independent Δchs1 showed no differences in the growth and production of sulfated polysaccharide compared to control. Phycoerythrin contents of the two independent mutants substantially increased regardless of light source. This study provides a novel applicability for the CRISPR/Cas9 RNP method in red microalgae toward a bio-product of interest. The obtained mutants could serve as potential producers of phycoerythrin if Porphyridium is selected as a natural source.

In Vivo Monitoring of Intracellular Metabolite in a Microalgal Cell Using an Aptamer/Graphene Oxide Nanosheet Complex.

Real-time sensing and imaging of intracellular metabolites in living cells are crucial tools for the characterization of complex biological processes, including the dynamic fluctuation of metabolites. Therefore, additional efforts are required to develop in vivo detection strategies for the visualization and quantification of specific target metabolites, particularly in microalgae. In this study, we developed a strategy to monitor a specific microalgal metabolite in living cells using an aptamer/graphene oxide nanosheet (GOnS) complex. As a proof-of-concept, ß-carotene, an antioxidant pigment that accumulates in most microalgal species, was chosen as a target metabolite. To achieve this, a ß-carotene-specific aptamer was selected through graphene oxide-assisted systematic evolution of ligands by exponential enrichment (GO-SELEX) and characterized thereafter. The aptamer could sensitively sense the changes in the concentration of ß-carotene (i.e., the target metabolite) and more specifically bind to ß-carotene than to nontargets. The selected aptamer was labeled with a fluorophore (fluorescein; FAM) and allowed to form an aptamer/GOnS complex that protected the aptamer from nucleic cleavages. The aptamer/GOnS complex was delivered into the cells via electroporation, thus enabling the sensitive monitoring of ß-carotene in the cell by quantifying the aptamer fluorescence intensity. The results suggest that our biocompatible strategy could be employed to visualize and semiquantify intracellular microalgae metabolites in vivo, which holds a great potential in diverse fields such as metabolite analysis and mutant screening.

Astaxanthin biosynthesis promotion with pH shock in the green microalga, Haematococcus lacustris.

In this study, the use of pH shock to improve astaxanthin synthesis in Haematococcus lacustris was investigated. It has been found that pH shock (pH = 4.5, 60 s) imposes stress in the cells and induces physiological changes, which result in astaxanthin accumulation. The optimal acid-base combination of pH shock was H2SO4-KOH, which increased the astaxanthin content per cell to 39 ± 6.92% than those of the control. In addition, pH shock can be applied simultaneously with the other inductive strategies such as high irradiance and carbon source supply. When high irradiance was applied simultaneously with pH shock, astaxanthin yield was increased 65 ± 0.541% than control. In addition, astaxanthin content per cell was increased 105 ± 6.66% than those of the control, with the concomitant application of carbon source addition with pH shock. Herein, these novel findings provide a useful technique for producing astaxanthin using H. lacustris.

Effect of Pseudoalteromonas sp. MEBiC 03485 on biomass production and sulfated polysaccharide biosynthesis in Porphyridium cruentum UTEX 161.

The effect of co-cultivation of Porphyridium cruentum UTEX 161 with Pseudoalteromonas sp. MEBiC 03485 on P. cruentum growth and its sulfated polysaccharide (EPS) production were examined. The strain MEBiC 03485 had beneficial effects on P. cruentum growth, EPS production, and EPS quality. These effects were due to a compound secreted by the strain MEBiC 03485. Notably, secretory compound treatment also increased intracellular phycoerythrin and phycocyanin content by 89.4% and 161%, respectively. In addition, the biological activities of EPS extracted from MEBiC 03485 treatment tended to be higher than the control without treatment. Our results suggest a novel approach for potentially enhancing the growth of P. cruentum and its EPS production and quality by co-culturing with the symbiotic strain MEBiC 03485.

Improvement of Euglena gracilis Paramylon Production through a Cocultivation Strategy with the Indole-3-Acetic Acid-Producing Bacterium Vibrio natriegens.

We investigated the putative effects on the growth and paramylon production of Euglena gracilis of cocultivation with Vibrio natriegensE. gracilis heterotrophically cocultivated with V. natriegens displayed significant increases in biomass productivity and paramylon content. In addition, the effects of the bacterial inoculum density and the timing of inoculation on the growth of E. gracilis were examined, to determine the optimal conditions for cocultivation. With the optimal deployment of V. natriegens, biomass productivity and paramylon content were increased by more than 20% and 35%, respectively, compared to those in axenic E. gracilis cultures. Interestingly, indole-3-acetic acid biosynthesized by V. natriegens was responsible for these enhancements of E. gracilis The morphology of cocultured E. gracilis cells was assessed. Paramylon granules extracted from the cocultivation were significantly larger than those from axenic culture. Our study showed that screening for appropriate bacteria and subsequent cocultivation with E. gracilis represented an effective way to enhance biomass and metabolite production.IMPORTANCEEuglena gracilis has attracted special interest due to its ability to excessively accumulate paramylon. Paramylon is a linear ß-1,3-glucan polysaccharide that is the principal polymer for energy storage in E. gracilis The polysaccharide features high bioactive functionality in the immune system. This study explored a new method to enhance the production of paramylon by E. gracilis, through cocultivation with the indole-3-acetic acid-producing bacterium Vibrio natriegens The enhanced production was achieved indirectly with the phytohormone-producing bacteria, instead of direct application of the hormone. The knowledge obtained in this study furthers the understanding of the effects of V. natriegens on the growth and physiology of E. gracilis.

Enhancement of growth and paramylon production of Euglena gracilis by co-cultivation with Pseudoalteromonas sp. MEBiC 03485.

This study investigated the putative effects of co-cultivation of Euglena gracilis with Pseudoalteromonas sp. MEBiC 03485 on the growth of E. gracilis and its paramylon production. The strain MEBiC 03485 had beneficial effects on the growth and paramylon contents of E. gracilis. To determine the optimal conditions for co-cultivation, the effects of algal to bacterial inoculum ratios and E. gracilis growth stages were examined. Under optimal conditions, the biomass productivity and paramylon production were increased by more than 23% and 34%, respectively. These effects were attributed to the extracellular polymeric substances (EPS) from the strain MEBiC 03485. GC-MS and HPAEC were carried out to analyze the composition of EPS. It was found that the EPS consisted of rhamnose, galactose, glucose, and mannose. These results suggest a novel approach for potentially enhancing the growth of E. gracilis as well as its paramylon production, via co-culturing with the symbiotic strain MEBiC 03485.

Enhancement of microalga Haematococcus pluvialis growth and astaxanthin production by electrical treatment.

In this study, we investigated the effects of electrical treatment on Haematococcus pluvialis growth. The slow growth of H. pluvialis is a major limitation for its mass production. We discovered that electrical treatment may promote the growth of H. pluvialis. To evaluate optimal growth-promoting conditions, the algal growth rate was investigated at various voltages. The optimum current was identified as 100 mA (voltage: 25 V). In comparison with the non-treated cells, those subjected to electrical treatment showed a 1.2 fold increase in cell density. Further experiments confirmed the direct impact of electrical treatment on the growth of H. pluvialis. The periodic application of electrical voltage resulted in a significant increase in the dry weight and astaxanthin production. The astaxanthin content in the periodic application of electrical treatment was 32.6 mg/L, which was a 10% increase compared to those in the non-treated controls. This strategy may serve as a novel approach to enhance H. pluvialis growth as well as astaxanthin production.

Adsorptive removal of harmful algal species Microcystis aeruginosa directly from aqueous solution using polyethylenimine coated polysulfone-biomass composite fiber.

In recent times, the treatment of harmful algal blooms (HABs) became an important environmental issue to preserve and remediate water resources globally. In the present study, the adsorptive removal of harmful algal species Microcystis aeruginosa directly from an aqueous medium was attempted. Waste biomass (Escherichia coli) was immobilized using polysulfone and coated using the cationic polymer polyethylenimine (PEI) to generate PEI-coated polysulfone-biomass composite fiber (PEI-PSBF). The density of M. aeruginosa in an aqueous medium (BG11) was significantly decreased by treatment with PEI-PSBF. additionally, analysis using FE-SEM, confirmed that the removal of M. aeruginosa algal cells by PEI-PSBF was caused by the adsorption mechanism. According to the profiles of phosphorus for the algal cell growth in M. aeruginosa cultivating samples, we found that the adsorbed M. aeruginosa onto the PEI-PSBF lost their biological activity compared to the non-treated M. aeruginosa cells.