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.

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.

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.

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.

The Ancient Phosphatidylinositol 3-Kinase Signaling System Is a Master Regulator of Energy and Carbon Metabolism in Algae.

Algae undergo a complete metabolic transformation under stress by arresting cell growth, inducing autophagy and hyper-accumulating biofuel precursors such as triacylglycerols and starch. However, the regulatory mechanisms behind this stress-induced transformation are still unclear. Here, we use biochemical, mutational, and "omics" approaches to demonstrate that PI3K signaling mediates the homeostasis of energy molecules and influences carbon metabolism in algae. In Chlamydomonas reinhardtii, the inhibition and knockdown (KD) of algal class III PI3K led to significantly decreased cell growth, altered cell morphology, and higher lipid and starch contents. Lipid profiling of wild-type and PI3K KD lines showed significantly reduced membrane lipid breakdown under nitrogen starvation (-N) in the KD. RNA-seq and network analyses showed that under -N conditions, the KD line carried out lipogenesis rather than lipid hydrolysis by initiating de novo fatty acid biosynthesis, which was supported by tricarboxylic acid cycle down-regulation and via acetyl-CoA synthesis from glycolysis. Remarkably, autophagic responses did not have primacy over inositide signaling in algae, unlike in mammals and vascular plants. The mutant displayed a fundamental shift in intracellular energy flux, analogous to that in tumor cells. The high free fatty acid levels and reduced mitochondrial ATP generation led to decreased cell viability. These results indicate that the PI3K signal transduction pathway is the metabolic gatekeeper restraining biofuel yields, thus maintaining fitness and viability under stress in algae. This study demonstrates the existence of homeostasis between starch and lipid synthesis controlled by lipid signaling in algae and expands our understanding of such processes, with biotechnological and evolutionary implications.

Influence of Water Depth on Microalgal Production, Biomass Harvest, and Energy Consumption in High Rate Algal Pond Using Municipal Wastewater.

The high rate algal ponds (HRAP) powered and mixed by a paddlewheel have been widely used for over 50 years to culture microalgae for the production of various products. Since light incidence is limited to the surface, water depth can affect microalgal growth in HRAP. To investigate the effect of water depth on microalgal growth, a mixed microalgal culture constituting three major strains of microalgae including Chlorella sp., Scenedesmus sp., and Stigeoclonium sp. (CSS), was grown at different water depths (20, 30, and 40 cm) in the HRAP, respectively. The HRAP with 20cm of water depth had about 38% higher biomass productivity per unit area (6.16 ± 0.33 g·m⁻²·d⁻¹) and required lower nutrients and energy consumption than the other water depths. Specifically, the algal biomass of HRAP under 20c m of water depth had higher settleability through larger floc size (83.6% settleability within 5 min). These results indicate that water depth can affect the harvesting process as well as cultivation of microalgae. Therefore, we conclude that water depth is an important parameter in HRAP design for mass cultivation of microalgae.

Application of high-salinity stress for enhancing the lipid productivity of Chlorella sorokiniana HS1 in a two-phase process.

Increased lipid accumulation of algal cells as a response to environmental stress factors attracted much attention of researchers to incorporate this stress response into industrial algal cultivation process with the aim of enhancing algal lipid productivity. This study applies high-salinity stress condition to a two-phase process in which microalgal cells are initially grown in freshwater medium until late exponential phase and subsequently subjected to high-salinity condition that induces excessive lipid accumulation. Our initial experiment revealed that the concentrated culture of Chlorella sorokiniana HS1 exhibited the intense fluorescence of Nile red at the NaCl concentration of 60 g/L along with 1 g/L of supplemental bicarbonate after 48 h of induction period without significantly compromising cultural integrity. These conditions were further verified with the algal culture grown for 7 days in a 1 L bottle reactor that reached late exponential phase; a 12% increment in the lipid content of harvested biomass was observed upon inducing high lipid accumulation in the concentrated algal culture at the density of 5.0 g DW/L. Although an increase in the sum of carbohydrate and lipid contents of harvested biomass indicated that the external carbon source supplemented during the induction period increased overall carbon assimilation, a decrease in carbohydrate content suggested the potential reallocation of cellular carbon that promoted lipid droplet formation under high-salinity stress. These results thus emphasize that the two-phase process can be successfully implemented to enhance algal lipid productivity by incorporating high-salinity stress conditions into the pre-concentrated sedimentation ponds of industrial algal production system.

Microalgal diversity fosters stable biomass productivity in open ponds treating wastewater.

It is established that biodiversity determines productivity of natural ecosystems globally. We have proved that abiotic factors influenced biomass productivity in engineered ecosystems i.e. high rate algal ponds (HRAPs), previously. This study demonstrates that biotic factors, particularly microalgal diversity, play an essential role in maintaining stable biomass productivity in HRAP treating municipal wastewater by mutualistic adaptation to environmental factors. The current study examined data from the second year of a two-year study on HRAP treating municipal wastewater. Microalgal diversity, wastewater characteristics, treatment efficiency and several environmental and meteorological factors were documented. Multivariate statistical analyses reveal that microalgae in uncontrolled HRAPs adapt to adverse environmental conditions by fostering diversity. Subsequently, five dominant microalgal strains by biovolume were isolated, enriched, and optimum conditions for high biomass productivity were ascertained. These laboratory experiments revealed that different microalgal strains dominate in different conditions and a consortium of these diverse taxa help in sustaining the algae community from environmental and predatory pressures. Diversity, niche or seasonal partitioning and mutualistic growth are pertinent in microalgal cultivation or wastewater treatment. Therefore, enrichment of selective species would deprive the collective adaptive ability of the consortium and encourage system vulnerability especially in wastewater treatment.

Influence of limiting factors on biomass and lipid productivities of axenic Chlorella vulgaris in photobioreactor under chemostat cultivation.

The understanding of process parameters and limiting conditions on microalgal biomass and lipid productivities is scarce especially in chemostat cultivation. In this study, the factors limiting growth of axenic Chlorella vulgaris OW-01 in cylindrical photobioreactor under chemostat cultivation were overcome in two phases. Physiological and physicochemical analyses determined inorganic carbon, phosphorous and light intensity as major limiting factors. Their effect on system productivity was ascertained and optimized in the first phase resulting in maximum biomass and lipid productivities of 538 and 128 (mg/L/d), respectively. In the second phase, the effect of dilution rate was evaluated under optimized conditions. The biomass and lipid productivities in this phase reached to 1013 and 270 (mg/L/d), respectively at a dilution rate of 0.75d(-1), yielding >10-fold cumulative increase in productivities. The study demonstrates addressing resource limitations by constant monitoring and optimization of chemostat cultivation to achieve high biomass and lipid productivities in photobioreactors.

Algae-bacteria interactions: Evolution, ecology and emerging applications.

Algae and bacteria have coexisted ever since the early stages of evolution. This coevolution has revolutionized life on earth in many aspects. Algae and bacteria together influence ecosystems as varied as deep seas to lichens and represent all conceivable modes of interactions - from mutualism to parasitism. Several studies have shown that algae and bacteria synergistically affect each other's physiology and metabolism, a classic case being algae-roseobacter interaction. These interactions are ubiquitous and define the primary productivity in most ecosystems. In recent years, algae have received much attention for industrial exploitation but their interaction with bacteria is often considered a contamination during commercialization. A few recent studies have shown that bacteria not only enhance algal growth but also help in flocculation, both essential processes in algal biotechnology. Hence, there is a need to understand these interactions from an evolutionary and ecological standpoint, and integrate this understanding for industrial use. Here we reflect on the diversity of such relationships and their associated mechanisms, as well as the habitats that they mutually influence. This review also outlines the role of these interactions in key evolutionary events such as endosymbiosis, besides their ecological role in biogeochemical cycles. Finally, we focus on extending such studies on algal-bacterial interactions to various environmental and bio-technological applications.

Organic carbon, influent microbial diversity and temperature strongly influence algal diversity and biomass in raceway ponds treating raw municipal wastewater.

Algae based wastewater treatment coupled to biofuel production has financial benefits and practical difficulties. This study evaluated the factors influencing diversity and growth of indigenous algal consortium cultivated on untreated municipal wastewater in a high rate algal pond (HRAP) for a period of 1 year using multivariate statistics. Diversity analyses revealed the presence of Chlorophyta, Cyanophyta and Bacillariophyta. Dominant microalgal genera by biovolume in various seasons were Scenedesmus sp., Microcystis sp., and Chlorella sp. Scenedesmus sp., persisted throughout the year but none of three strains co-dominated with the other. The most significant factors affecting genus dominance were temperature, inflow cyanophyta and organic carbon concentration. Cyanophyta concentration affected microalgal biomass and diversity, whereas temperature impacted biomass. Preferred diversity of microalgae is not sustained in wastewater systems but is obligatory for biofuel production. This study serves as a guideline to sustain desired microalgal consortium in wastewater treatment plants for biofuel production.

Enhancing microalgal biomass productivity by engineering a microalgal-bacterial community.

This study demonstrates that ecologically engineered bacterial consortium could enhance microalgal biomass and lipid productivities through carbon exchange. Phycosphere bacterial diversity analysis in xenic Chlorella vulgaris (XCV) confirmed the presence of growth enhancing and inhibiting microorganisms. Co-cultivation of axenic C. vulgaris (ACV) with four different growth enhancing bacteria revealed a symbiotic relationship with each bacterium. An artificial microalgal-bacterial consortium (AMBC) constituting these four bacteria and ACV showed that the bacterial consortium exerted a statistically significant (P<0.05) growth enhancement on ACV. Moreover, AMBC had superior flocculation efficiency, lipid content and quality. Studies on carbon exchange revealed that bacteria in AMBC might utilize fixed organic carbon released by microalgae, and in return, supply inorganic and low molecular weight (LMW) organic carbon influencing algal growth and metabolism. Such exchanges, although species specific, have enormous significance in carbon cycle and can be exploitated by microalgal biotechnology industry.

A cost analysis of microalgal biomass and biodiesel production in open raceways treating municipal wastewater and under optimum light wavelength.

Open raceway ponds are cost-efficient for mass cultivation of microalgae compared with photobioreactors. Although low-cost options like wastewater as nutrient source is studied to overcome the commercialization threshold for biodiesel production from microalgae, a cost analysis on the use of wastewater and other incremental increases in productivity has not been elucidated. We determined the effect of using wastewater and wavelength filters on microalgal productivity. Experimental results were then fitted into a model, and cost analysis was performed in comparison with control raceways. Three different microalgal strains, Chlorella vulgaris AG10032, Chlorella sp. JK2, and Scenedesmus sp. JK10, were tested for nutrient removal under different light wavelengths (blue, green, red, and white) using filters in batch cultivation. Blue wavelength showed an average of 27% higher nutrient removal and at least 42% higher chemical oxygen demand removal compared with white light. Naturally, the specific growth rate of microalgae cultivated under blue wavelength was on average 10.8% higher than white wavelength. Similarly, lipid productivity was highest in blue wavelength, at least 46.8% higher than white wavelength, whereas FAME composition revealed a mild increase in oleic and palmitic acid levels. Cost analysis reveals that raceways treating wastewater and using monochromatic wavelength would decrease costs from 2.71 to 0.73 $/kg biomass. We prove that increasing both biomass and lipid productivity is possible through cost-effective approaches, thereby accelerating the commercialization of low-value products from microalgae, like biodiesel.

Nutrient removal and biofuel production in high rate algal pond using real municipal wastewater.

This study evaluated the growth and nutrient removal ability of an indigenous algal consortium on real untreated municipal wastewater in a high rate algal pond (HRAP). The HRAP was operated semicontinuously under different hydraulic retention times (HRT: 2, 4, 6, and 8 days). The average removal efficiencies of chemical oxygen demand, and total nitrogen and phosphate of real municipal wastewater were maintained at 85.44 ± 5.10%, 92.74 ± 5.82%, and 82.85 ± 8.63%, respectively, in 2 day HRT. Algae dominated the consortium and showed high settling efficiency (99%), and biomass and lipid productivity of 0.500 ± 0.03 g/l/day and 0.103 ± 0.0083 g/l/day (2 day HRT), respectively. Fatty acid methyl ester analysis revealed a predominance of palmitate (C16:0), palmitoleate (C16:1), linoleate (C18:2), and linolenate (C18:3). Microalgal diversity analyses determined the presence of Chlorella, Scenedesmus, and Stigeoclonium as the dominant microalgae. The algal consortium provides significant value not only in terms of energy savings and nutrient removal but also because of its bioenergy potential as indicated by the lipid content (20-23%) and FAME profiling.

Microalgae-associated bacteria play a key role in the flocculation of Chlorella vulgaris.

Flocculation is most preferred method for harvesting microalgae, however, the role of bacteria in microalgal flocculation process is still unknown. This study proves that bacteria play a profound role in flocculating by increasing the floc size resulting in sedimentation of microalgae. A flocculating activity of 94% was achieved with xenic Chlorella vulgaris culture as compared to 2% achieved with axenic culture. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene of xenic C. vulgaris culture revealed the presence of Flavobacterium sp., Terrimonas sp., Sphingobacterium sp., Rhizobium sp. and Hyphomonas sp. as microalgae-associated bacteria. However when Flavobacterium, Terrimonas, Sphingobacterium were eliminated by fluorescence activated cell sorter (FACS), flocculating activity reduced to 3%. Further studies with cell free extracts also suggest that bacterial extracellular substances might also have a role in enhancing flocculation. We conclude that the collective presence of certain bacteria is the determining factor in flocculation of C. vulgaris.

Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii.

Phenotypic and genotypic changes in Chlamydomonas reinhardtii BafJ5, a starchless mutant, with respect to lipid metabolism was studied in different trophic states under nitrogen (N) sufficient and limited conditions. Interestingly, cellular lipid content increased linearly with input acetate concentration with highest lipid content (∼42%) under nitrogen limitation and mixotrophic state. RT-qPCR studies indicate that key fatty acid biosynthesis genes are down-regulated under N limitation but not under mixotrophic state, whereas, ACS2, encoding Acetyl-CoA synthetase, and DGTT4, encoding Diacylglycerol O-acyltransferase, are up-regulated under all conditions. These results collectively indicate that acetate is the limiting factor and central molecule in lipid droplet synthesis. The study also provides further evidence of the presence of a chloroplast pathway for triacylglycerol synthesis in microalgae.

Novel approach for the development of axenic microalgal cultures from environmental samples.

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence-activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.

Simple, rapid and cost-effective method for high quality nucleic acids extraction from different strains of Botryococcus braunii.

This study deals with an effective nucleic acids extraction method from various strains of Botryococcus braunii which possesses an extensive extracellular matrix. A method combining freeze/thaw and bead-beating with heterogeneous diameter of silica/zirconia beads was optimized to isolate DNA and RNA from microalgae, especially from B. braunii. Eukaryotic Microalgal Nucleic Acids Extraction (EMNE) method developed in this study showed at least 300 times higher DNA yield in all strains of B. braunii with high integrity and 50 times reduced working volume compared to commercially available DNA extraction kits. High quality RNA was also extracted using this method and more than two times the yield compared to existing methods. Real-time experiments confirmed the quality and quantity of the input DNA and RNA extracted using EMNE method. The method was also applied to other eukaryotic microalgae, such as diatoms, Chlamydomonas sp., Chlorella sp., and Scenedesmus sp. resulting in higher efficiencies. Cost-effectiveness analysis of DNA extraction by various methods revealed that EMNE method was superior to commercial kits and other reported methods by >15%. This method would immensely contribute to area of microalgal genomics.

Bio-mitigation of CO(2), calcite formation and simultaneous biodiesel precursors production using Chlorella sp.

In this study, an attempt was made to use micro-algal system for the production of biodiesel precursors and simultaneous CO(2) mitigation. Chlorella sp. was found to have a higher growth rate as compared to the other algal species tested namely Chlamydomonas sp. and Synnecococcus sp. At different CO(2) concentrations (0.03%, 3%, 10% and 15%), the lipid productivity was 23.0, 20.0 and 27.3mg/L/d respectively. Calcite produced was characterized using FT-IR, SEM and XRD. The FAME in crude biofuel was analyzed by GC-FID that found to contain palmitic acid (C16:0), docosapentaenoic acid (C22:5) and docosahexaenoic acid (C22:6). The calorific value of Chlorella sp. was found to be 29kJ/g which is higher than values reported for fresh water microalgae making it a potential candidate to be used as an alternate fuel.