Unlocking synergies: Harnessing the potential of biological methane sequestration through metabolic coupling between Methylomicrobium alcaliphilum 20Z and Chlorella sp. HS2.

A halotolerant consortium between microalgae and methanotrophic bacteria could effectively remediate in situ CH4 and CO2, particularly using saline wastewater sources. Herein, Methylomicrobium alcaliphilum 20Z was demonstrated to form a mutualistic association with Chlorella sp. HS2 at a salinity level above 3.0%. Co-culture significantly enhanced the growth of both microbes, independent of initial inoculum ratios. Additionally, increased methane provision in enclosed serum bottles led to saturated methane removal. Subsequent analyses suggested nearly an order of magnitude increase in the amount of carbon sequestered in biomass in methane-fed co-cultures, conditions that also maintained a suitable cultural pH suitable for methanotrophic growth. Collectively, these results suggest a robust metabolic coupling between the two microbes and the influence of the factors other than gaseous exchange on the assembled consortium. Therefore, multi-faceted investigations are needed to harness the significant methane removal potential of the identified halotolerant consortium under conditions relevant to real-world operation scenarios.

Cultivation of Chlorella sp. HS2 using wastewater from soy sauce factory.

Incorporation of wastewater from industrial sectors into the design of microalgal biorefineries has significant potential for advancing the practical application of this emerging industry. This study tested various food industrial wastewaters to assess their suitability for microalgal cultivation. Among these wastewaters, defective soy sauce (DSS) and soy sauce wastewater (SWW) were chosen but DSS exhibited the highest nutrient content with 13,500 ppm total nitrogen and 3051 ppm total phosphorus. After diluting DSS by a factor of 50, small-scale cultivation of microalgae was conducted to optimize culture conditions. SWW exhibited optimal growth at 25-30 °C and 300-500 µE m-2 s-1, while DSS showed optimal growth at 30-35 °C. Based on a 100-mL lab-scale and 3-L outdoor cultivation with an extended cultivation period, DSS outperformed SWW, exhibiting higher final biomass productivity. Additionally, nutrient-concentrated nature of DSS is advantageous for transportation at an industrial scale, leading us to select it as the most promising feedstock for microalgal cultivation. With further optimization, DSS has the potential to serve as an effective microalgal cultivation feedstock for large-scale biomass production.

Identification of Loliolide with Anti-Aging Properties from Scenedesmus deserticola JD052.

Herein, different extracts of Scenedesmus deserticola JD052, a green microalga, were evaluated in vitro as a potential anti-aging bioagent. Although post-treatment of microalgal culture with either UV irradiation or high light illumination did not lead to a substantial difference in the effectiveness of microalgal extracts as a potential anti-UV agent, the results indicated the presence of a highly potent compound in ethyl acetate extract with more than 20% increase in the cellular viability of normal human dermal fibroblasts (nHDFs) compared with the negative control amended with DMSO. The subsequent fractionation of the ethyl acetate extract led to two bioactive fractions with high anti-UV property; one of the fractions was further separated down to a single compound. While electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy analysis identified this single compound as loliolide, its identification has been rarely reported in microalgae previously, prompting thorough systematic investigations into this novel compound for the nascent microalgal industry.

7S,15R-Dihydroxy-16S,17S-epoxy-docosapentaenoic Acid Overcomes Chemoresistance of 5-Fluorouracil by Suppressing the Infiltration of Tumor-Associated Macrophages and Inhibiting the Activation of Cancer Stem Cells in a Colorectal Cancer Xenograft Model.

Although the tumor bulk is initially reduced by 5-fluorouracil (5-FU), chemoresistance developed due to prolonged chemotherapy in colorectal cancer (CRC). The enrichment of cancer stem cells (CSCs) and the infiltration of tumor-associated macrophages (TAMs) contribute to chemoresistance and poor outcomes. A docosahexaenoic acid derivative developed by our group, 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), exerts antitumor effects against TAMs infiltration and CSCs enrichment in our previous study. The current study aimed to investigate whether diHEP-DPA was able to overcome chemoresistance to 5-FU in CRCs, together with the potential synergistic mechanisms in a CT26-BALB/c mouse model. Our results suggested that although 5-FU inhibited tumor growth, 5-FU enriched CSCs via the WNT/ß-catenin signaling pathway, resulting in chemoresistance in CRCs. However, we revealed that 5-FU promoted the infiltration of TAMs via the NF-kB signaling pathway and improved epithelial-mesenchymal transition (EMT) via the signal transducer and activator of the transcription 3 (STAT3) signaling pathway; these traits were believed to contribute to CSC activation. Furthermore, supplementation with diHEP-DPA could overcome drug resistance by decreasing the CSCs, suppressing the infiltration of TAMs, and inhibiting EMT progression. Additionally, the combinatorial treatment of diHEP-DPA and 5-FU effectively enhanced phagocytosis by blocking the CD47/signal regulatory protein alpha (SIRPα) axis. These findings present that diHEP-DPA is a potential therapeutic supplement to improve drug outcomes and suppress chemoresistance associated with the current 5-FU-based therapies for colorectal cancer.

Effects of Fish Oil, Lipid Mediators, Derived from Docosahexaenoic Acid, and Their Co-Treatment against Lipid Metabolism Dysfunction and Inflammation in HFD Mice and HepG2 Cells.

Although fish oil (FO) and lipid mediators (LM) derived from polyunsaturated fatty acids can prevent obesity, their combined effects and cellular metabolism remain unclear. Therefore, this study aimed to examine the potential protective and metabolic effects of FO in combination with LM (a mixture of 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX [3:47:50], derived from docosahexaenoic acid (DHA)) on palmitic acid (PA)-induced HepG2 cells and high-fat- diet (HFD)-induced C57BL/6J mice after 9-week treatment. Lipid metabolism disorders and inflammation induced by HFD and PA were substantially reduced after FO and LM treatment. Further, FO and LM treatments reduced lipid accumulation by increasing fatty acid oxidation via peroxisome proliferator-activated receptor α and carnitine-palmitoyl transferase 1 as well as by decreasing fatty acid synthesis via sterol regulatory element-binding protein-1c and fatty acid synthase. Finally, FO and LM treatment reduced inflammation by blocking the NF-κB signaling pathway. Importantly, the combination of FO and LM exhibited more robust efficacy against nonalcoholic fatty liver disease, suggesting that FO supplemented with LM is a beneficial dietary strategy for treating this disease.

How do freshwater microalgae and cyanobacteria respond to antibiotics?

Antibiotic pollution is an emerging environmental challenge. Residual antibiotics from various sources, including municipal and industrial wastewater, sewage discharges, and agricultural runoff, are continuously released into freshwater environments, turning them into reservoirs that contribute to the development and spread of antibiotic resistance. Thus, it is essential to understand the impacts of antibiotic residues on aquatic organisms, especially microalgae and cyanobacteria, due to their crucial roles as primary producers in the ecosystem. This review summarizes the effects of antibiotics on major biological processes in freshwater microalgae and cyanobacteria, including photosynthesis, oxidative stress, and the metabolism of macromolecules. Their adaptive mechanisms to antibiotics exposure, such as biodegradation, bioadsorption, and bioaccumulation, are also discussed. Moreover, this review highlights the important factors affecting the antibiotic removal pathways by these organisms, which will promote the use of microalgae-based technology for the removal of antibiotics. Finally, we offer some perspectives on the opportunities for further studies and applications.

Gross Negligence: Impacts of Microplastics and Plastic Leachates on Phytoplankton Community and Ecosystem Dynamics.

Plastic debris is an established environmental menace affecting aquatic systems globally. Recently, microplastics (MP) and plastic leachates (PL) have been detected in vital human organs, the vascular system, and in vitro animal studies positing severe health hazards. MP and PL have been found in every conceivable aquatic ecosystem─from open oceans and deep sea floors to supposedly pristine glacier lakes and snow covered mountain catchment sites. Many studies have documented the MP and PL impacts on a variety of aquatic organisms, whereby some exclusively focus on aquatic microorganisms. Yet, the specific MP and PL impacts on primary producers have not been systematically analyzed. Therefore, this review focuses on the threats posed by MP, PL, and associated chemicals on phytoplankton, their comprehensive impacts at organismal, community, and ecosystem scales, and their endogenous amelioration. Studies on MP- and PL-impacted individual phytoplankton species reveal the production of reactive oxygen species, lipid peroxidation, physical damage of thylakoids, and other physiological and metabolic changes, followed by homo- and heteroaggregations, ultimately eventuating in decreased photosynthesis and primary productivity. Likewise, analyses of the microbial community in the plastisphere show a radically different profile compared to the surrounding planktonic diversity. The plastisphere also enriches multidrug-resistant bacteria, cyanotoxins, and pollutants, accelerating microbial succession, changing the microbiome, and thus, affecting phytoplankton diversity and evolution. These impacts on cellular and community scales manifest in changed ecosystem dynamics with widespread bottom-up and top-down effects on aquatic biodiversity and food web interactions. These adverse effects─through altered nutrient cycling─have "knock-on" impacts on biogeochemical cycles and greenhouse gases. Consequently, these impacts affect provisioning and regulating ecosystem services. Our citation network analyses (CNA) further demonstrate dire effects of MP and PL on all trophic levels, thereby unsettling ecosystem stability and services. CNA points to several emerging nodes indicating combined toxicity of MP, PL, and their associated hazards on phytoplankton. Taken together, our study shows that ecotoxicity of plastic particles and their leachates have placed primary producers and some aquatic ecosystems in peril.

The emerging potential of natural and synthetic algae-based microbiomes for heavy metal removal and recovery from wastewaters.

Heavy Metal (HM) bioremoval by microbes is a successful, environment-friendly technique, particularly at low concentrations of HMs. Studies using algae, bacteria, and fungi reveal promising capabilities in isolation and when used in consortia. Yet, few reviews have emphasized individual and collective HM removal rates and the associated mechanisms in natural or synthetic microbiomes. Besides discussing the limitations of conventional and synthetic biology approaches, this review underscores the utility of indigenous microbial taxon, i.e., algae, fungi, and bacteria, in HM removal with adsorption capacities and their synergistic role in microbiome-led studies. The detoxification mechanisms studied for certain HMs indicate distinctive removal pathways in each taxon which points to an enhanced effect when used as a microbiome. The role and higher efficacies of the designer microbiomes with complementing and mutualistic taxa are also considered, followed by recovery options for a circular bioeconomy. The citation network analysis further validates the multi-metal removal ability of microbiomes and the restricted capabilities of the individual counterparts. In precis, the study reemphasizes increased metal removal efficiencies of inter-taxon microbiomes and the mechanisms for synergistic and improved removal, eventually drawing attention to the benefits of ecological engineering approaches compared to other alternatives.

Extra benefit of microalgae in raw piggery wastewater treatment: pathogen reduction.

BACKGROUND: Monitoring microbial communities especially focused on pathogens in newly developed wastewater treatment systems is recommended for public health. Thus, we investigated the microbial community shift in a pilot-scale microalgal treatment system for piggery wastewater. RESULTS: Microalgae showed reasonable removal efficiencies for COD and ammonia, resulting in higher transparency of the final effluent. Metagenome and microbial diversity analyses showed that heterotrophic microalgal cultivation barely changed the bacterial community; however, the mixotrophic microalgal cultivation induced a sudden change. In addition, an evaluation of risk groups (RGs) of bacteria showed that raw piggery wastewater included abundant pathogens, and the microalgal treatment of the raw piggery wastewater decreased the RG2 pathogens by 63%. However, co-cultivation of microalgae and the most dominant RG2 pathogen, Oligella, showed no direct effects between them. CONCLUSIONS: Thus, a microbial interaction network was constructed to elucidate algae-bacteria interrelationships, and the decrease in Oligella was indirectly connected with microalgal growth via Brevundimonas, Sphingopyxis, and Stenotrophomonas. In a validation test, 3 among 4 connecting bacterial strains exhibited inhibition zones against Oligella. Therefore, we showed that microalgal wastewater treatment causes a decrease in RG2 bacteria, which is an indirect impact of microalgae associated with bacteria. Video abstract.

Algal biopolymers as sustainable resources for a net-zero carbon bioeconomy.

The era for eco-friendly polymers was ushered by the marine plastic menace and with the discovery of emerging pollutants such as micro-, nano-plastics, and plastic leachates from fossil fuel-based polymers. This review investigates algae-derived natural, carbon neutral polysaccharides and polyesters, their structure, biosynthetic mechanisms, biopolymers and biocomposites production process, followed by biodegradability of the polymers. The review proposes acceleration of research in this promising area to address the need for eco-friendly polymers and to increase the cost-effectiveness of algal biorefineries by coupling biofuel, high-value products, and biopolymer production using waste and wastewater-grown algal biomass. Such a strategy improves overall sustainability by lowering costs and carbon emissions in algal biorefineries, eventually contributing towards the much touted circular, net-zero carbon future economies. Finally, this review analyses the evolution of citation networks, which in turn highlight the emergence of a new frontier of sustainable polymers from algae.

Development of a novel nannochloropsis strain with enhanced violaxanthin yield for large-scale production.

BACKGROUND: Nannochloropsis is a marine microalga that has been extensively studied. The major carotenoid produced by this group of microalgae is violaxanthin, which exhibits anti-inflammatory, anti-photoaging, and antiproliferative activities. Therefore, it has a wide range of potential applications. However, large-scale production of this pigment has not been much studied, thereby limiting its industrial application. RESULTS: To develop a novel strain producing high amount of violaxanthin, various Nannochloropsis species were isolated from seawater samples and their violaxanthin production potential were compared. Of the strains tested, N. oceanica WS-1 exhibited the highest violaxanthin productivity; to further enhance the violaxanthin yield of WS-1, we performed gamma-ray-mediated random mutagenesis followed by colorimetric screening. As a result, Mutant M1 was selected because of its significant higher violaxanthin content and biomass productivity than WS-1 (5.21 ± 0.33 mg g- 1 and 0.2101 g L- 1 d- 1, respectively). Subsequently, we employed a 10 L-scale bioreactor to confirm the large-scale production potential of M1, and the results indicated a 43.54 % increase in violaxanthin production compared with WS-1. In addition, comparative transcriptomic analysis performed under normal light condition identified possible mechanisms associated with remediating photo-inhibitory damage and other key responses in M1, which seemed to at least partially explain enhanced violaxanthin content and delayed growth. CONCLUSIONS: Nannochloropsis oceanica mutant (M1) with enhanced violaxanthin content was developed and its physiological characteristics were investigated. In addition, enhanced production of violaxanthin was demonstrated in the large-scale cultivation. Key transcriptomic responses that are seemingly associated with different physiological responses of M1 were elucidated under normal light condition, the details of which would guide ongoing efforts to further maximize the industrial potential of violaxanthin producing strains.

Biodegradable films based on chitosan and defatted Chlorella biomass: Functional and physical characterization.

Biodegradable films based on chitosan, glycerol, and defatted Chlorella biomass (DCB) were prepared and characterized in terms of thermal stability, mechanical, water barrier, and optical properties. Increasing DCB content from 5 to 25 wt% increased tensile strength of chitosan films by 235%. The incorporation of DCB decreased both moisture content and swelling degree of chitosan/defatted Chlorella biomass (Cs/DCB) films. Furthermore, increasing the content of defatted algal biomass decreased light transmission and reduced water vapor permeability of composite films by more than 60%. As confirmed by scanning electron microscopy and Fourier transform infrared analysis, such improvement in functional and physical properties is mainly due to the homogeneous and uniform distribution of DCB into the polymeric matrix along with the establishment of strong hydrogen bond interactions between chitosan and algal biomass constituents. Moreover, Cs/DCB composite films showed more than 50% of degradation in 60 days soil burial test.

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.

Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in green microalgae.

BACKGROUND: For decades, plastic has been a valuable global product due to its convenience and low price. For example, polyethylene terephthalate (PET) was one of the most popular materials for disposable bottles due to its beneficial properties, namely impact resistance, high clarity, and light weight. Increasing demand of plastic resulted in indiscriminate disposal by consumers, causing severe accumulation of plastic wastes. Because of this, scientists have made great efforts to find a way to biologically treat plastic wastes. As a result, a novel plastic degradation enzyme, PETase, which can hydrolyze PET, was discovered in Ideonella sakaiensis 201-F6 in 2016. RESULTS: A green algae, Chlamydomonas reinhardtii, which produces PETase, was developed for this study. Two representative strains (C. reinhardtii CC-124 and CC-503) were examined, and we found that CC-124 could express PETase well. To verify the catalytic activity of PETase produced by C. reinhardtii, cell lysate of the transformant and PET samples were co-incubated at 30 °C for up to 4 weeks. After incubation, terephthalic acid (TPA), i.e. the fully-degraded form of PET, was detected by high performance liquid chromatography analysis. Additionally, morphological changes, such as holes and dents on the surface of PET film, were observed using scanning electron microscopy. CONCLUSIONS: A PET hydrolyzing enzyme, PETase, was successfully expressed in C. reinhardtii, and its catalytic activity was demonstrated. To the best of our knowledge, this is the first case of PETase expression in green algae.

Biocompatible liquid-type carbon nanodots (C-paints) as light delivery materials for cell growth and astaxanthin induction of Haematococcus pluvialis.

In this study, we aimed to demonstrate the feasibility of the application of biocompatible liquid type fluorescent carbon nanodots (C-paints) to microalgae by improving microalgae productivity. C-paints were prepared by a simple process of ultrasound irradiation using polyethylene glycol (PEG) as a passivation agent. The resulting C-paints exhibited a carbonyl-rich surface with good uniformity of particle size, excellent water solubility, photo-stability, fluorescence efficiency, and good biocompatibility (<10.0 mg mL-1 of C-paints concentration). In the practical application of C-paints to microalgae culture, the most effective and optimized condition leading to growth promoting effect was observed at a C-paints concentration of 1.0 mg mL-1 (>20% higher than the control cell content). A C-paints concentration of 1-10.0 mg mL-1 induced an approximately >1.8 times higher astaxanthin content than the control cells. The high light delivery effect of non-cytotoxic C-paints was applied as a stress condition for H. pluvialis growth and was found to play a major role in enhancing productivity. Notably, the results from this study are an essential approach to improve astaxanthin production, which can be used in various applications because of its therapeutic effects such as cancer prevention, anti-inflammation, immune stimulation, and treatment of muscle-soreness.

Author Correction: Application of biosurfactant from Bacillus subtilis C9 for controlling cladoceran grazers in algal cultivation systems.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Monoterpenoid Loliolide Regulates Hair Follicle Inductivity of Human Dermal Papilla Cells by Activating the Akt/ß-Catenin Signaling Pathway.

Loliolide is one of the most ubiquitous monoterpenoid compounds found in algae, and its potential therapeutic effect on various dermatological conditions via agent-induced biological functions, including anti-oxidative and anti-apoptotic properties, was demonstrated. Here, we investigated the effects of loliolide on hair growth in dermal papilla (DP) cells, the main components regulating hair growth and loss conditions. For this purpose, we used a threedimensional (3D) DP spheroid model that mimics the in vivo hair follicle system. Biochemical assays showed that low doses of loliolide increased the viability and size of 3D DP spheroids in a dose-dependent manner. This result correlated with increases in expression levels of hair growth-related autocrine factors including VEGF, IGF-1, and KGF. Immunoblotting and luciferase-reporter assays further revealed that loliolide induced AKT phosphorylation, and this effect led to stabilization of ß-catenin, which plays a crucial role in the hair-inductive properties of DP cells. Further experiments showed that loliolide increased the expression levels of the DP signature genes, ALP, BMP2, VCAN, and HEY1. Furthermore, conditioned media from loliolide-treated DP spheroids significantly enhanced proliferation and the expression of hair growth regulatory genes in keratinocytes. These results suggested that loliolide could function in the hair growth inductivity of DP cells via the AKT/ ß-catenin signaling pathways.

Dynamic Interactions between Autophagosomes and Lipid Droplets in Chlamydomonas reinhardtii.

Autophagy is a highly conserved catabolic process in eukaryotic cells by which waste cellular components are recycled to maintain growth in both favorable and stress conditions. Autophagy has been linked to lipid metabolism in microalgae; however, the mechanism underlying this interaction remains unclear. In this study, transgenic Chlamydomonas reinhardtii cells that stably express the red fluorescent protein (mCherry) tagged-ATG8 as an autophagy marker were established. By using this tool, we were able to follow the autophagy process in live microalgal cells under various conditions. Live-cell and transmission electron microscopy (TEM) imaging revealed physical contacts between lipid droplets and autophagic structures during the early stage of nitrogen starvation, while fusion of these two organelles was observed in prolonged nutritional deficiency, suggesting that an autophagy-related pathway might be involved in lipid droplet turnover in this alga. Our results thus shed light on the interplay between autophagy and lipid metabolism in C. reinhardtii, and this autophagy marker would be a valuable asset for further investigations on autophagic processes in microalgae.