Green and efficient method to acquire high-value phycobiliprotein from microalgal biomass involving deep eutectic solvent-based ultrasound-assisted extraction.

Phycoerythrin (PE) is a phycobiliprotein holding great potential as a high-value food colorant and medicine. Deep eutectic solvent (DES)-based ultrasound-assisted extraction (UAE) was applied to extract B-PE by disrupting the resistant polysaccharide cell wall of Porphyridium purpureum. The solubility of cell wall monomers in 31 DESs was predicted using COSMO-RS. Five glycerol-based DESs were tested for extraction, all of which showed significantly higher B-PE yields by up to 13.5 folds than water. The DES-dependent B-PE extraction efficiencies were proposedly associated with different cell disrupting capabilities and protein stabilizing effects of DESs. The DES-based UAE method could be considered green according to a metric assessment tool, AGREEprep. The crude extract containing DES was further subjected to aqueous two-phase system, two-step ammonium sulfate precipitation, and ultrafiltration processes. The final purified B-PE had a PE purity ratio of 3.60 and a PC purity ratio of 0.08, comparable to the purity of commercial products.

Chitosan-modified cotton fiber: An efficient and reusable adsorbent in removal of harmful cyanobacteria, Microcystis aeruginosa from aqueous phases.

In the present study, to remove harmful cyanobacterial species Microcystis aeruginosa from aqueous phases, adsorption-based strategy was utilized. For this strategy, the surface of cotton fiber was modified using chitosan molecules to develop a highly efficient and ecofriendly adsorbent in removal of Microcystis aeruginosa from aqueous solution. The pristine cotton fiber could not remove M. aeruginosa, while the chitosan-modified cotton (CS-m-Cotton) showed the 95% of cell removal efficiency within 12 h. The surface characteristics of chitosan-modified cotton compared to the pristine cotton fiber was examined by various surface analysis methods. In addition, the pre-treatment of pristine cotton using sodium hydroxide solution was an important factor for enhancement of chitosan modification efficiency on the cotton fiber. The developed chitosan-modified cotton fiber could be reusable for M. aeruginosa cell removal after the simple desorption treatment using ultrasonication in alkaline solution. During the repeated adsorbent regeneration and reuse, the chitosan-modified cotton maintained its M. aeruginosa removal efficiencies (>90%). From the acute toxicity assessment using the chitosan-modified cotton and, the measurements of chemical oxygen demand and microcystin level changes in the M. aeruginosa treatment process using the adsorbent, the environmental safety of the adsorption strategy using the developed adsorbent could be confirmed. Based on our results, the chitosan-modified cotton fiber could be proposed as an efficient and ecofriendly solution for remediation of harmful cyanobacterial species occurring water resources.

Effects of micro-sized biodegradable plastics on Microcystis aeruginosa.

Plethora of plastics are being used in current society, generating huge amounts of plastic waste. Non-biodegradability of conventional plastics is one of the main challenges to treat plastic waste. In an effort to increase the efficiency of plastic waste treatment, biodegradable plastics have gained attention. Although the use of biodegradable plastics has been increased, their potential effects on the environments are not fully elucidated yet. In this study, the impacts of micro-sized non-biodegradable plastic (i.e., polystyrene (PS)) and micro-sized biodegradable plastics (i.e., polycaprolactone (PCL) and polylactic acid (PLA)) on Microcystis aeruginosa were investigated. Regardless of microplastic (MP) types, MP treatments inhibited the growth of M. aeruginosa at the beginning (4 days) while significant dose-dependent effect was not observed in the range of 0.1 to 10 mg/L. However, after long-term exposure (12 days), micro-sized biodegradable plastics stimulated the growth of M. aeruginosa (up to 73 % increase compared to the control). The photosynthetic activity showed a similar trend to the cell growth. The MP treatments induced the production of extracellular polymeric substances (EPS). Indeed, micro-sized PCL and PLA stimulated the production of protein compounds in EPS. These might have affected the releases of chemicals from PCL and PLA, suggesting that the chemicals in biodegradable plastic leachates would promote the growth of M. aeruginosa in long-term exposure. The MP treatments also induced cyanotoxin (microcystin-LR) productions. Our results give a new insight into the cyanobacterial blooming and suggest a novel relationship between harmful algal blooms (HABs) and biodegradable plastics.

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance.

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

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.

Ultrasonic Treatment Enhanced Astaxanthin Production of Haematococcus pluvialis.

In this study, effects of ultrasonic treatment on Haematococcus pluvialis (H. pluvialis) were investigated. It has been confirmed that the ultrasonic stimulation acted as stress resources in the red cyst stage H. pluvialis cells containing astaxanthin, resulting in additional astaxanthin production. With the increase in production of astaxanthin, the average diameter of H. pluvialis cells increased accordingly. In addition, to determine how ultrasonic stimulation had an effect on the further biosynthesis of astaxanthin, genes related to astaxanthin synthesis and cellular ROS level were measured. As a result, it was confirmed that astaxanthin biosynthesis related genes and cellular ROS levels were increased, and thus ultrasonic stimulation acts as an oxidative stimulus. These results support the notion on the effect of the ultrasonic treatment, and we believe our novel approach based on the ultrasonic treatment would help to enhance the astaxanthin production from H. pluvialis.

Bacteroides faecium sp. nov. isolated from human faeces.

A novel bacterial strain, CBA7301T, was isolated from human faeces and was characterised using a polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequences revealed that CBA7301T represented a member of the genus Bacteroides, in the family Bacteroidaceae. The similarity between the 16S rRNA gene sequence of CBA7301T and that of its most closely related species, Bacteroides faecichinchillae JCM 17102T, was 96.2 %, and the average nucleotide identity between these two strains was 77.9 %. The genome size was 6 782 182 bp, and the DNA G+C content was 42.5 mol%. Cells of CBA7301T were Gram-stain-negative, strictly anaerobic and rod-shaped. The optimal growth of this organism occurred at 30-35 °C, pH 7.0 and 0.5 % (w/v) NaCl. The respiratory quinone was menaquinone 10. The predominant polar lipids were phosphatidylethanolamine, phospholipids and aminophospholipids. The major cellular fatty acid was anteiso-C15 : 0. According to the results of the polyphasic taxonomic analysis, CBA7301T represents a novel species of the genus Bacteroides, which we named Bacteroides faecium sp. nov. The type strain is CBA7301T (=KCCM 43355T=ATCC TSD-227T).

The cytotoxicity of nano- and micro-sized graphene oxides on microalgae depends on the characteristics of cell wall and flagella.

Cytotoxic effects of emerging contaminants in aquatic environments have been widely studied using diverse microalgal species. However, the role of microalgal characteristics such as presence/absence of cell wall or flagella on cytotoxicity of contaminants was not elucidated yet. In this study, four different Chlamydomonas reinhardtii strains that have different characteristics were used to confirm how these characteristics affect toxicity of contaminants, nano-/micro-sized graphene oxide (GO). The nano-sized GO inhibited the growth of cell wall-deficient strains and reduced the photosynthetic activity. The micro-sized GO inhibited the growth of all strains, but the inhibition efficiency was higher in flagella-deficient strains, indicating that cell wall and flagella have different roles in response to contaminant exposure. The electron microscopy analysis demonstrated that nano-sized GO caused the cell rupture in cell wall-deficient strains. In flagella-deficient strains, the nano- and micro-sized GOs were parallelly attached on the surface of cells, covering the cells. The wrapping of flagella-deficient cells by GO led to the increase of reactive oxygen species (ROS) contents. These results indicate main cytotoxic mechanism of nano-sized GO was the membrane damage of cells, and the presence of cell wall can protect the cells from the attack of nano-sized GO. On the one hand, the presence of flagella might help to avoid the attachment of GO while the cell proliferation and photosynthesis were inhibited in flagella-deficient cells due to the GO wrapping. Overall, given that different microalgal species have different characteristics and these characteristics might affect the cytotoxic effect of the contaminants, it is of great importance to consider the characteristics of test microalgal species when evaluating the cytotoxic mechanism of the nano-/micro-sized pollutants.

Volatile fatty acid-treated mixotrophic cultivation of lipid/carbohydrate-rich cyanobacterial species, Pseudanabaena mucicola GO0704, for the enhancement of biofuel production.

Cyanobacteria-derived biofuels can be helpful in achieving a circular bioeconomy. To increase the production of biodiesel/bioethanol from cyanobacterium, Pseudanabaena mucicola GO0704, mixotrophic cultivation using volatile fatty acid (VFA), a cheap organic carbon source, was performed. The treatment of butyric acid or acetic acid enhanced the cell growth, particularly, the dry weight of the butyric acid-treated cells was 2.30-fold higher than the control. The enhancement of the growth led to the increase of metabolite (i.e., lipid and carbohydrate) productions, resulting in high amount of biodiesel and bioethanol to be produced. Butyric acid was more effective compared to acetic acid and the productions of biodiesel (52.2 mg/L) and bioethanol (132.6 mg/L) from the butyric acid-treated P. mucicola GO0704 were 2.34- and 2.17-fold higher compared to the control, respectively. This study will provide a foundation to commercialize the cyanobacteria-based carbon-neutral fuels, and ultimately, achieve a circular bioeconomy.

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.

Mixotrophic Cultivation of a Native Cyanobacterium, Pseudanabaena mucicola GO0704, to Produce Phycobiliprotein and Biodiesel.

Global warming has accelerated in recent decades due to the continuous consumption of petroleum-based fuels. Cyanobacteria-derived biofuels are a promising carbon-neutral alternative to fossil fuels that may help achieve a cleaner environment. Here, we propose an effective strategy based on the large-scale cultivation of a newly isolated cyanobacterial strain to produce phycobiliprotein and biodiesel, thus demonstrating the potential commercial applicability of the isolated microalgal strain. A native cyanobacterium was isolated from Goryeong, Korea, and identified as Pseudanabaena mucicola GO0704 through 16s RNA analysis. The potential exploitation of P. mucicola GO0704 was explored by analyzing several parameters for mixotrophic culture, and optimal growth was achieved through the addition of sodium acetate (1 g/l) to the BG-11 medium. Next, the cultures were scaled up to a stirred-tank bioreactor in mixotrophic conditions to maximize the productivity of biomass and metabolites. The biomass, phycobiliprotein, and fatty acids concentrations in sodium acetate-treated cells were enhanced, and the highest biodiesel productivity (8.1 mg/l/d) was achieved at 96 h. Finally, the properties of the fuel derived from P. mucicola GO0704 were estimated with converted biodiesels according to the composition of fatty acids. Most of the characteristics of the final product, except for the cloud point, were compliant with international biodiesel standards [ASTM 6761 (US) and EN 14214 (Europe)].

Strategies to control the growth of cyanobacteria and recovery using adsorption and desorption.

In this study, adsorption strategy using diethylenetriamine-modified cotton fiber (DETA-cotton) was investigated to control the target cells in aqueous phase. Adsorptive removal of M. aeruginosa using the DETA-cotton showed decrease in cell concentration from (100 ± 4.0) × 104 cells/mL to (32.1 ± 0.7) × 104 cells/mL in 24 h, and the concentration of microcystin did not increase during the removal process. Also, an increase in the amine groups on the surface was confirmed through the surface characterization by FT-IR and XPS. Desorption process was performed to analyze total lipid and fatty acid contents for potential use as bio-energy resources. About 90 % of the adsorbed cells were recovered through desorption, and the lipid content and composition were more suitable for use as biodiesel raw materials. Our adsorption-based approach might provide feasible solution not only to counteract environmental issue HABs but also to recover energy-resources from the harmful cyanobacterial species.

Elucidating the mechanisms underlying the cytotoxic effects of nano-/micro-sized graphene oxide on the microalgae by comparing the physiological and morphological changes in different trophic modes.

Understanding the cytotoxic mechanisms of environmental contaminants is important to estimate their environmental impacts and prepare guidelines for pollution control. Many studies have assessed the cytotoxic mechanisms of graphene oxide (GO), an emerging aquatic contaminant. However, in many cases, the effect of GO size and putative trophic modes of microalgae on cytotoxicity has been neglected, hindering complete understanding of the cytotoxic mechanisms of GO. In this study, the microalga Euglena gracilis cultivated under light (phototrophic) or dark (heterotrophic) conditions was exposed to two sizes of GO [nano-sized (N) and micro-sized (M)] for assessing the effect of GO on microalgal growth. The cytotoxic effect of GO was higher under phototrophic conditions than under heterotrophic conditions, suggesting that a major cytotoxic mechanism of GO is related to photosynthetic activity inhibition. Moreover, N-GO showed higher toxicity than M-GO. The morphological and physiological changes in N-GO- and M-GO-exposed E. gracilis were assessed to further elucidate the cytotoxic mechanisms. N-GO internalized the cells via endocytic activity/piercing, whereas M-GO partially attached to the cell surface and did not enter the cells. Moreover, N-GO and M-GO negatively affected the cells by inducing oxidative stress; the oxidative stress parameters were higher in N-GO-exposed cells than in M-GO-exposed cells.

Role of combinated lactic acid bacteria in bacterial, viral, and metabolite dynamics during fermentation of vegetable food, kimchi.

Lactic acid bacteria (LAB) in kimchi, a traditional Korean food, are major fermentative microorganisms affecting the quality, safety, and nutritional and organoleptic properties of the final product. In this study, we determined the role of three key LAB strains, Leuconostoc gelidum, Latilactobacillus sakei, Weissella koreensis originated from different raw ingredients during natural fermentation, as opposed to an axenic environment. Starter cultures were inoculated into food with wild indigenous microbial communities, and the dynamics of bacterial communities and metabolites were analyzed during fermentation. As bacteriophages within the food viral community directly affect fermentation by influencing bacterial function and composition, the diversity and composition of DNA viral communities were compared with those of corresponding bacterial communities using a metagenomic approach. Our results provide insights into the ecological role of LAB starters in food fermentation and the potential impact of bacteriophages as modulators of bacterial communities associated with the fermentation properties of kimchi.

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.

Amination of Non-Functional Polyvinyl Chloride Polymer Using Polyethyleneimine for Removal of Phosphorus from Aqueous Solution.

The eutrophication of freshwater environments caused by an excess inflow of phosphorus has become a serious environmental issue because it is a crucial factor for the occurrence of harmful algal blooms (HABs) in essential water resources. The adsorptive removal of phosphorus from discharged phosphorus containing effluents has been recognized as one of the most promising solutions in the prevention of eutrophication. In the present study, a polyvinyl chloride (PVC)-polyethyleneimine (PEI) composite fiber (PEI-PVC) was suggested as a stable and recoverable adsorbent for the removal of phosphorus from aqueous phases. The newly introduced amine groups of the PEI-PVC were confirmed by a comparison between the FT-IR and XPS results of the PVC and PEI-PVC. The phosphorus sorption on the PEI-PVC was pH dependent. At the optimum pH for phosphorus adsorption (pH 5), the maximum adsorption capacity of the PEI-PVC fiber was estimated to be 11.2 times higher (19.66 ± 0.82 mg/g) than that of conventional activated carbon (1.75 ± 0.4 mg/g) using the Langmuir isotherm model. The phosphorus adsorption equilibrium of the PEI-PVC was reached within 30 min at pH 5. From the phosphorus-loaded PEI-PVC, 97.4% of the adsorbed amount of phosphorus on the PEI-PVC could be recovered by employing a desorption process using 1M HCl solution without sorbent destruction. The regenerated PEI-PVC through the desorption process maintained a phosphorus sorption capacity almost equal to that of the first use. In addition, consistently with the PVC fiber, the PEI-PVC fiber did not elute any toxic chlorines into the solution during light irradiation. Based on these results, the PEI-PVC fiber can be suggested as a feasible and stable adsorbent for phosphorus removal.

Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

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.

Fluorogenic "on-off" nanosensor based on dual-quenching effect for imaging intracellular metabolite of various microalgae.

Sensing intracellular compounds such as ATP in living microalgal cells is of great importance in diverse fields. To achieve this, nanosensing platform composed of graphene oxide (GO) and ATP aptamer (APT) was applied to diverse microalgal cells (Chlamydomonas reinhardtii, Chlorella vulgaris, Anabaena flos-aquae, and Ochromonas danica). The nanosized GO was characterized with atomic force microscopy (AFM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The nanosensing platform (GO-APT) was prepared by attaching fluorophore-labeled APT to GO. GO-APT was applicable to only cell wall-deficient species (O. danica and mutant strains of C. reinhardtii) and the existence of flagella did not affect the uptake of the GO-APT by the cells. These results indicate that the cell wall is the primary barrier of GO-APT internalization for sensing application. To reduce the background fluorescence signal elicited by nonspecific displacement of the fluorophore-labeled probe, APT was modified as molecular beacon (MB) type (APTMB). Owing to the double quenching effect (by GO and quencher-labeled complementary sequence), the background signal significantly reduced. Cytotoxicity of GO-APTMB on the microalgal species was also tested. The application of GO-APTMB had no effect on the growth of microalgae. Given that diverse aptamer sequences had been screened, the sensing platform is not limited for detecting ATP only, but also can be applied to other metabolite imaging by simply changing the aptamer sequences. Our research will contribute to broadening the application of GO and aptamer beacon complex for intracellular metabolite imaging and detecting.

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.