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Haematococcus lacustris is a ubiquitous unicellular green alga with industrial bioproduct applications, namely, as feedstock for natural astaxanthin. We report the annotated 291.5 Mbp genome for H. lacustris Liv1 to support future algal research in the areas of carotenoid biosynthesis and crop protection.
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The overarching goal of this work was to accelerate the commercialization of high productivity, mixed community microalgal treatment technologies for the synergistic treatment of wastewater and the production of biofuel feedstocks. This project addressed a critical barrier to the financial viability and energy efficiency of algal wastewater treatment: an inability to design and operate high-rate processes that reliably achieve target effluent qualities, areal productivities, and biochemical compositions (lipid, protein, carbohydrate content) despite fluctuations in wastewater composition, weather, and microbial communities. Key outcomes from this work include an optimized and controlled Advanced Biological Nutrient Recovery (ABNR) design as well as a suite of open-source tools that include a calibrated and validated algae process simulator in QSDsan and a novel low-cost, real-time microbial monitoring tool. These tools can be leveraged by other algal cultivation and wastewater treatment technology developers in future work.
The terrestrial green alga Chromochloris zofingiensis is an emerging model species with potential applications including production of triacylglycerol or astaxanthin. How C. zofingiensis interacts with the diverse substrates during trophic transitions is unknown. To characterize its substrate utilization and secretion dynamics, we cultivated the alga in a soil-based defined medium in transition between conditions with and without glucose supplementation. Then, we examined its exometabolite and endometabolite profiles. This analysis revealed that regardless of trophic modes, C. zofingiensis preferentially uptakes exogenous lysine, arginine, and purines, while secreting orotic acid. Here, we obtained metabolomic evidences that C. zofingiensis may use arginine for putrescine synthesis when in transition to heterotrophy, and for the TCA cycle during transition to photoautotrophy. We also report that glucose and fructose most effectively inhibited photosynthesis among thirteen different sugars. The utilized or secreted metabolites identified in this study provide important information to improve C. zofingiensis cultivation, and to expand its potential industrial and pharmaceutical applications.
Polyurethane is a versatile material with a wide range of applications in the production of foam products such as car seats, mattresses, footwear, insulation foam, and more. The traditional process for producing polyurethane foam involves the fast reaction of isocyanate with water, which generates CO2 as a blowing agent. However, the use of isocyanate has raised concerns due to its potential for causing allergic reactions and aspiration symptoms. This has led to increased regulation of isocyanate, leading to a growing interest in developing alternatives. Non-isocyanate polyurethane (NIPU) has emerged as a promising alternative to traditional toxic PU production. The most promising approach for producing NIPU is the aminolysis of cyclic carbonate with diamine. The NIPU produced through this process has a polyhydroxyurethane structure, which is similar to traditional PU chemistry. We have demonstrated that microalgal oil is a favored feedstock, as it can provide high reactivity for industrial applications. However, the NIPU production process does not generate gas, and a blowing agent is required to achieve the desired foam structure. One of the challenges in producing NIPU foam is to match the bubble formation and curing rates. We have developed a novel approach to producing NIPU foam that utilizes carbamate as a bifunctional blowing and crosslinking agent. We have used vegetable oil and microalgal oil as feedstocks to produce various NIPU for characterization. The development of microalgal NIPU provides an exciting opportunity for reducing the environmental impact of foam production and improving the safety of the materials used in the production process. The production of value-added NIPU foam can also drive down the cost of microalgal biorefinery, advancing the commercialization of microalgal biofuel.
Large scale commercial cultivation of microalgae year-round is limited by seasonal stress conditions. The rapid growth and high CO 2 capture of the marine microalga Picochlorum celeri is largely inhibited under winter stress conditions of low temperature and high light. Herein, we demonstrated a nanotechnology approach to enhance the biomass productivity and CO 2 capture of P. celeri under abiotic stress by interfacing with antioxidant cerium oxide nanozymes (nanoceria). Antioxidant nanoceria catalytically scavenged reactive oxygen species (ROS) generated under stress conditions, reducing damage to the microalgae photosynthetic machinery in chloroplasts. Negatively charged poly-acrylic acid-coated nanoceria (PNC, 10 μM) were biocompatible in microalgae cells and colocalized with chloroplasts. In contrast, positively charged aminated nanoceria (ANC) resulted in microalgae aggregation (>50 μM) and were toxic at all concentrations tested (≥10 μM). PNC reduction of ROS levels in microalgae (78%) and superoxide levels (26%), enhanced microalgae growth (65%), photosynthetic performance (130%), and CO 2 uptake rate (380%) under low-temperature stress (15 °C) and high light (500 μmol/m 2 /s PAR) stress relative to controls without nanoceria. Nanoceria augmentation of microalgae provides a rapid and facile technology to increase algae CO 2 capture and biomass under stress conditions.
ABSTRACT To combat the increasing levels of carbon dioxide (CO 2 ) released from the combustion of fossil fuels, microalgae have emerged as a promising strategy for biological carbon capture, utilization, and storage. This study used a marine microalgal strain, Nannochloropsis oceanica IMET1, which thrives in high CO 2 concentrations. A high-pH, high-alkalinity culture was designed for CO 2 capture through algal biomass production as well as permanent sequestration through calcium carbonate (CaCO 3 ) precipitation. This was accomplished by timed pH elevation and the addition of sodium bicarbonate to cultures of N. oceanica grown at lab scale (1 L) and pilot scale (500 L) with 10% and 5% CO 2 , respectively. Our data showed that 0.02 M NaHCO 3 promoted algal growth and that sparging cultures with ambient air after 12 days raised pH and created favorable CaCO 3 formation conditions. At the 1 L scale, we reached 1.52 g L −1 biomass after 12 days and an extra 9.3% CO 2 was captured in the form of CaCO 3 precipitates. At the 500 L pilot scale, an extra 60% CO 2 was captured (Day 40) with a maximum CO 2 capture rate of 63.2 g m −2 day −1 (Day 35). Bacterial communities associated with the microalgae were dominated by two novel Patescibacteria. Functional analysis revealed that genes for several plant growth-promotion traits (PGPTs) were enriched within this group. The microalgal-bacterial coculture system offers advantages for enhanced carbon mitigation through biomass production and simultaneous precipitation of recalcitrant CaCO 3 for long-term CO 2 storage. IMPORTANCE Capturing carbon dioxide (CO 2 ) released from fossil fuel combustion is of the utmost importance as the impacts of climate change continue to worsen. Microalgae can remove CO 2 through their natural photosynthetic pathways and are additionally able to convert CO 2 into a stable, recalcitrant form as calcium carbonate (CaCO 3 ). We demonstrate that microalgae-based carbon capture systems can be greatly improved with high pH and high alkalinity by providing optimal conditions for carbonate precipitation. Our results with the microalga, Nannochloropsis oceanica strain IMET1, show an extra 9.3% CO 2 captured as CaCO 3 at the 1 L scale and an extra 60% CO 2 captured at the 500 L (pilot) scale. Our optimized system provides a novel approach to capture CO 2 through two mechanisms: (i) as organic carbon within microalgal biomass and (ii) as inorganic carbon stored permanently in the form of CaCO 3.
Microalgae offer a compelling platform for the production of commodity products, due to their superior photosynthetic efficiency, adaptability to nonarable lands and nonpotable water, and their capacity to produce a versatile array of bioproducts, including biofuels and biomaterials. However, the scalability of microalgae as a bioresource has been hindered by challenges such as costly biomass production related to vulnerability to pond crashes during large-scale cultivation. This study presents a pipeline for the genetic engineering and pilot-scale production of biodiesel and thermoplastic polyurethane precursors in the extremophile species Chlamydomonas pacifica. This extremophile microalga exhibits exceptional resilience to high pH (>11.5), high salinity (up to 2% NaCl), and elevated temperatures (up to 42 °C). Initially, we evolved this strain to also have a high tolerance to high light intensity (>2000 μE/m 2 /s) through mutagenesis, breeding, and selection. We subsequently genetically engineered C. pacifica to significantly enhance lipid production by 28% and starch accumulation by 27%, all without affecting its growth rate. We demonstrated the scalability of these engineered strains by cultivating them in pilot-scale raceway ponds and converting the resulting biomass into biodiesel and thermoplastic polyurethanes. This study showcases the complete cycle of transforming a newly discovered species into a commercially relevant commodity production strain. This research underscores the potential of extremophile algae, including C. pacifica, as a key species for the burgeoning sustainable bioeconomy, offering a viable path forward in mitigating environmental challenges and supporting global bioproduct demands.
Microalgae are increasingly recognized for their potential in wastewater treatment and the sustainable production of feedstock for fuel, feed, food, and other bioproducts. Like conventional agricultural systems, algal cultivation involves complex microbial communities. However, despite their pivotal role in cultivation outcomes, especially at the commodity-scale, the critical interactions between microalgae and their microbiomes are often overlooked. Here we synthesize current knowledge on the taxonomic diversity, ecological roles, and biotechnological potential of algal microbiomes, with a focus on their interactions with algal hosts through nutrient exchange, growth modulation, pathogen defense, and environmental conditioning. We also examine how environmental factors such as nutrient availability, salinity, and temperature influence these interactions. Advances in microbiome engineering, including synthetic biology and ecological approaches, offer opportunities to enhance beneficial algal-microbiome interactions, thereby improving growth, resilience, and yield. These advancements could lead to more sustainable and economically viable microalgae cultivation, with far-reaching implications for environmental management and biotechnological innovation. By addressing key economic and environmental barriers, microbiome engineering holds transformative potential to revolutionize large-scale algae cultivation and provide sustainable solutions to global challenges.
Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for ~50% of global photosynthesis, compartmentalize their Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase (Rubisco) into a specialized structure known as the pyrenoid when the cells experience limiting CO 2 conditions; this compartmentalization is a component of the CO 2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO 2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO 2 limitation, although a role for this reorganization in CCM function remains unclear. We used the green microalga Chlamydomonas reinhardtii to monitor changes in mitochondrial position and ultrastructure as cells transition between high CO 2 and Low/Very Low CO 2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral cell location and orient in parallel tubular arrays that extend along the cell’s apico-basal axis. We show that these ultrastructural changes correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membranes, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, with the latter involved in membrane–microtubule interactions. Furthermore, blocking mitochondrial respiration in VLC-acclimated cells reduces the affinity of the cells for Ci. Overall, our results suggest that mitochondrial repositioning functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.
Abstract Plastic pollution has become a global crisis, with microplastics contaminating every environment on the planet, including our food, water, and even our bodies. In response, there is a growing interest in developing plastics that biodegrade naturally, thus avoiding the creation of persistent microplastics. As a mechanism to increase the rate of polyester plastic degradation, we examined the potential of using the green microalgaChlamydomonas reinhardtiifor the expression and secretion of PHL7, an enzyme that breaks down post-consumer polyethylene terephthalate (PET) plastics. We engineeredC. reinhardtiito secrete active PHL7 enzyme and selected strains showing robust expression, by using agar plates containing a polyester polyurethane (PU) dispersion as an efficient screening tool. This method demonstrated the enzyme’s efficacy in degrading ester bond-containing plastics, such as PET and bio-based polyurethanes, and highlights the potential for microalgae to be implemented in environmental biotechnology. The effectiveness of algal-expressed PHL7 in degrading plastics was shown by incubating PET with the supernatant from engineered strains, resulting in substantial plastic degradation, confirmed by mass spectrometry analysis of terephthalic acid formation from PET. Our findings demonstrate the feasibility of polyester plastic recycling using microalgae to produce plastic-degrading enzymes. This eco-friendly approach can support global efforts toward eliminating plastic in our environment, and aligns with the pursuit of low-carbon materials, as these engineered algae can also produce plastic monomer precursors. Finally, this data demonstratesC. reinhardtiicapabilities for recombinant enzyme production and secretion, offering a “green” alternative to traditional industrial enzyme production methods.
AbstractPlastic pollution has become a global crisis, with microplastics contaminating every environment on the planet, including our food, water, and even our bodies. In response, there is a growing interest in developing plastics that biodegrade naturally, thus avoiding the creation of persistent microplastics. As a mechanism to increase the rate of polyester plastic degradation, we examined the potential of using the green microalgaChlamydomonas reinhardtiifor the expression and secretion of PHL7, an enzyme that breaks down post-consumer polyethylene terephthalate (PET) plastics. We engineeredC. reinhardtiito secrete active PHL7 enzyme and selected strains showing robust expression, by using agar plates containing a polyester polyurethane (PU) dispersion as an efficient screening tool. This method demonstrated the enzyme’s efficacy in degrading ester bond-containing plastics, such as PET and bio-based polyurethanes, and highlights the potential for microalgae to be implemented in environmental biotechnology. The effectiveness of algal-expressed PHL7 in degrading plastics was shown by incubating PET with the supernatant from engineered strains, resulting in substantial plastic degradation, confirmed by mass spectrometry analysis of terephthalic acid (TPA) formation from PET. Our findings demonstrate the feasibility of polyester plastic recycling using microalgae to produce plastic-degrading enzymes. This eco-friendly approach can support global efforts toward eliminating plastic in our environment, and aligns with the pursuit of low-carbon materials, as these engineered algae can also produce plastic monomer precursors. Finally, this data demonstratesC. reinhardtiicapabilities for recombinant enzyme production and secretion, offering a “green” alternative to traditional industrial enzyme production methods.Graphical Abstract
Phytoplankton respond to their environment through genetic adaptation and plasticity to maintain fitness. This poses challenges when growing microalgae for industrial applications because, inherently, outdoor mass cultivation may lead to adaptations that alter desirable phenotypic traits and environmental niches. Here, we used common garden experiments to quantify the plasticity and adaptive responses to seasonal and geographic climate variation of Nannochloropsis, a microalga commonly used in biotechnology. An initially monoclonal strain was grown outdoors across four locations in Hawaii, Texas, California, and New Mexico. Following 17 and 22 months of cultivation outdoors, we collected samples during winter and summer, respectively, and we compared strains' growth from the four sites across temperature and light gradients in the laboratory. Despite hundreds of generations of exposure to divergent climates, with ~20°C and three-fold differences in daily light intensity, strains showed only minor differences in performance. Thermal performance varied more among seasons than sites, whereas light performance varied with both season and site. Our study indicates that Nannochloropsis exhibits broad plasticity in response to light and temperature, which may inhibit genetic adaptation in space or time. Highly variable field conditions, with daily and seasonal climate fluctuations, may favor plasticity and prevent the rapid adaptation often seen in laboratory studies of microorganisms in constant environments.
ABSTRACT Cobamides, the vitamin B 12 (cobalamin) family of cofactors, are used by most organisms but produced by only a fraction of prokaryotes, and are thus considered key shared nutrients among microbes. Cobamides are structurally diverse, with multiple different cobamides found in most microbial communities. The ability to use different cobamides has been tested for several bacteria and microalgae, and nearly all show preferences for certain cobamides. This approach is limited by the commercial unavailability of cobamides other than cobalamin. Here, we have extracted and purified seven commercially unavailable cobamides to characterize bacterial cobamide preferences based on growth in specific cobamide-dependent conditions. The tested bacteria include engineered strains of Escherichia coli , Sinorhizobium meliloti , and Bacillus subtilis expressing native or heterologous cobamide-dependent enzymes, cultured under conditions that functionally isolate specific cobamide-dependent processes such as methionine synthesis. Comparison of these results to those of previous studies of diverse bacteria and microalgae revealed that a broad diversity of cobamide preferences exists not only across different organisms but also between different cobamide-dependent metabolic pathways within the same organism. The microbes differed in the cobamides that support growth most efficiently, cobamides that do not support growth, and the minimum cobamide concentrations required for growth. The latter differ by up to four orders of magnitude across organisms from different environments and by up to 20-fold between cobamide-dependent enzymes within the same organism. Given that cobamides are shared, required for use of specific growth substrates, and essential for central metabolism in certain organisms, cobamide preferences likely impact community structure and function. IMPORTANCE Nearly all bacteria are found in microbial communities with tens to thousands of other species. Molecular interactions such as metabolic cooperation and competition are key factors underlying community assembly and structure. Cobamides, the vitamin B 12 family of enzyme cofactors, are one such class of nutrients, produced by only a minority of prokaryotes but required by most microbes. A unique aspect of cobamides is their broad diversity, with nearly 20 structural forms identified in nature. Importantly, this structural diversity impacts growth as most bacteria that have been tested show preferences for specific cobamide forms. We measured cobamide-dependent growth in several model bacteria and compared the results to those of previous analyses of cobamide preference. We found that cobamide preferences vary widely across bacteria, showing the importance of characterizing these aspects of cobamide biology to understand the impact of cobamides on microbial communities.
Microalgae cultivation processes exist only for a few species used in commercial applications. Their domestication is still in its infancy, with major advances required, specifically to overcome limiting factors in microalgae production. The goal is to develop superior strains without the need of genetic technologies that would require lengthy regulatory permits, and often limit consumer acceptance. Adaptive Laboratory Evolution techniques, alone or in conjunction with sexual recombination, can allow for rapid develop of improved strains and their industrial production. Approaches include increased photon utilization efficiency by cultures operating under full sunlight conditions due to higher light saturation levels, allowing for higher productivities under outdoor conditions. Decades of research yielded some promising results under controlled conditions with a few specific mutant strains. However, these failed to achieve the anticipated higher productivities in actual algal mass cultures, in part due to the inability of mutations to overcome photoinhibition, reactive oxygen species, and other pleiotropic impacts on photosynthesis.
Filamentous algae, which grow in long, hair-like filaments within biofilms, play a crucial role in wastewater treatment due to their ability to produce significant biomass and their resistance to predation compared to traditional microalgal treatments. These algae can effectively uptake and utilize pollutants, particularly excessive nitrogen (ammonia, nitrate, nitrite) and phosphorus (phosphate), making filamentous algae valuable for wastewater treatment, as well as bioethanol and biodiesel production due to high lipid productions. However, each algal species possesses different capacities, necessitating a thorough genetic identification and understanding of each community. A major challenge in accurately assessing these communities is the lack of coverage in large sequencing databases which can lead to misrepresentation of the true composition and abundance of organisms and overall sequencing bias. To address this, I evaluated chemical and physical techniques for separating filamentous algae from mixed biofilms to achieve clean genetic sequencing results. I employed pH washing (0.001M HCl, 0.001M HCl, DiH2O, 0.0001M HCl, 0.001M HCl) for chemical treatment, followed by physical separation through centrifugation (5000rpm, 6500rpm) or filtration (2mm, 250um, 75um). The most successful method was deionized water washing, which yielded clear differences across stacked filters; the 2mm filtrate showed high levels of filamentous algae, with microalgae eluting in the 75um filtrate or remaining within agglutinations of algae larger filters. Base washing eluted the highest concentrations of microalgae, with larger filter sizes retaining more filamentous algae, indicating the breakdown of extracellular polymeric substances (EPS). Our downstream plans include sending the high-throughput next-generation sequencing to confirm the purity and ratios of filamentous and non-filamentous algae, as well as bacteria present, thereby validating the success of our treatments. Potential applications include creating community-based fractions for analysis, refining current sequencing data with clearer isolations, and generating designer biofilms to enhance our understanding of community interactions.
This study investigated the potential for a synthetic consortium of mutualistic terrestrial microbes— comprising the fungi Laccaria bicolor and Serendipita indica alongside bacterial Pseudomonas strains—to influence the growth and productivity of the freshwater microalgae Chlorella vulgaris. The project aimed to determine if microbial complexes engineered to enhance terrestrial plant growth could provide similar growth-promoting benefits or pathogen resistance within an aquatic algal system. Using a quantitative experimental design, C. vulgaris was co-cultured with the microbial mix under controlled laboratory conditions, with growth rates, biomass density, and metabolic activity monitored over a standard cultivation period. The results demonstrated no significant symbiotic relationship or growth enhancement between these terrestrial microbes and the microalgae, as the C. vulgaris maintained independent growth trajectories unaffected by the fungal or bacterial inoculants. We conclude that the specialized mutualisms of these fungi and bacteria are likely niche-specific to vascular plants and do not readily translate to the phycosphere of C. vulgaris. These findings are valuable to synthetic biologists and bioenergy researchers, as they define the functional boundaries of inter-kingdom microbial engineering and underscore the necessity of selecting niche-compatible species when designing consortia for industrial algal cultivation.
Growing interest in sustainable food and biofuel research has necessitated high quality axenic oleaginous microalgal strains. Unfortunately, most strains available in culture banks contain commensal microbes such as bacteria and the default decontamination method involves antibiotic treatment which has begun to exacerbate the emergence of antibiotic resistance. To overcome this problem, anoxic photosensitisation was investigated as an alternate approach. Four oleaginous microalgal species (Tetradesmus obliquus, Desmodesmus armatus, Chlorella vulgaris and Nannochloropsis limnetica) were incubated in varying concentrations of Rose Bengal (0 μM, 1 μM, 3 μM or 9 μM) either in normal (oxic) or anoxic conditions, for 72 h under light (8.85 ± 0.4 W/m 2 ) in a specially designed heterotrophic growth complex (HGC) medium, followed by 72 h in standard Bold's Basal Medium (BBM). Commonly used antibiotics-based protocol was used as the control method. Post treatment, cell numbers and percentage populations were counted with Flow Cytometry, and viability was tested using standard plating methods using BBM and LB. Additionally, the contaminating microbes in the cultures were profiled using 16Ss rRNA sequencing. Anoxic conditions were able to significantly decrease bacterial content, albeit with an equally detrimental effect on the microalgal population. Although the responses differed between the microalgae, anoxic incubation along with Rose Bengal at 3 μM was able to completely decontaminate N. limnetica and C. vulgaris, while D. armatus and T. obliquus could be decontaminated with an additional streak-plating step. None of the cultures could be decontaminated using antibiotics treatment. These results suggest that axenisation of microalgal cultures was largely due to anoxy, that was synergistically enhanced by Rose Bengal at a concentration of ≥3 μM.