Effects of stepwise changes in dissolved carbon dioxide concentrations on metabolic activity in Chlorella for spaceflight applications
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Emily E. Matula↗
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Metabolic response of Chlorella vulgaris to a transient thermal environment for supporting simultaneous air revitalization and thermal control in a crewed habitat
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Emily E. Matula↗
DISCOVR strain pipeline screening – Part II: Winter and summer season areal productivities and biomass compositional shifts in climate-simulation photobioreactor cultures
Assessing the seasonal biomass productivity and compositional shift dynamics under simulated outdoor culture conditions of the top 22 algae strains selected during Tier I flask screening is an important step in the further prioritization of strains with regard to outdoor pond cultivation. These top 22 strains were subjected to Tier II testing in the PNNL Laboratory Environmental Algae Pond Simulator (LEAPS) photobioreactors, simulating light and temperature conditions of 20 cm deep outdoor ponds during the Arizona winter and summer season. All strains were grown in two consecutive nutrient-replete batch culture experiments at their particular optimal medium salinity to quantify their respective seasonal linear-phase areal biomass productivities. To determine biomass compositional shifts in response to nutrient-depletion, the LEAPS cultures were allowed to enter a 9-day nutrient depletion phase at the end of the second batch run. The following strains were evaluated in winter-season climate-simulated cultures and are listed in the order from highest (7.9 g m -2 day -1 ) to lowest (2.3 g m -2 day -1 ) areal N-replete biomass productivity: Monoraphidium minutum 26B-AM, Tetraselmis striata LANL1001, Chlorella vulgaris LRB AZ-1201, Micractinium reisseri NREL14-F2, Monoraphidium sp. MONOR1, Chlorella vulgaris NREL4-C12, Scenedesmus obliquus UTEX393, Scenedesmus acutus LRB-AP-0401, Nannochloropsis oceanica CCAP849/10, and Stichococcus minutus CCALA727. The following strains were evaluated in summer-season climate-simulated cultures and are listed in the order from highest (31.8 g m -2 day -1 ) to lowest (14.2 g m -2 day -1 ) areal N-replete biomass productivity: Picochlorum renovo NREL39-A8, Scenedesmus obliquus UTEX393, Porphyridium cruentum CCMP675, Picochlorum celeri TG2-WT-CSM/EMRE, Chlorella sorokiniana DOE1116, Stichococcus minor CCMP819, Picochlorum oklahomensis CCMP2329, Chlorella sorokiniana DOE1412 (UTEXB3016), Scenedesmus rubescens NREL46B-D3, Picochlorum soloecismus DOE101, Tetraselmis striata LANL1001, Scenedesmus obliquus DOE 0152.z, and Agmenellum quadruplicatum UTEX2268. All LEAPS cultures experienced a significant reduction in areal biomass productivity in response to nutrient-depletion, from 7-16% in the winter season simulation and up to 1-60% in the summer season simulation. Finally, for 10 of the strains tested, the carbohydrate content more than doubled upon nutrient depletion, and for 9 strains, the lipid content increased by over 50% of the initial content.
09 BIOMASS FUELS↗
DISCOVR strain pipeline screening – Part I: Maximum specific growth rate as a function of temperature and salinity for 38 candidate microalgae for biofuels production
Here, to identify high productivity strains for microalgal biofuels generation, the maximum specific growth rate of 38 strains was measured as a function of salinity (i.e., 5, 15, and 35 PSU) and temperature (i.e., at 8 temperatures along a linear gradient from ca. 5 to 45°C) to determine the most suitable growth medium salinity and best growing season, respectively, for outdoor raceway pond cultivation. The following strains were evaluated: Agmenellum quadruplicatum UTEX 2268, Anabaena sp. ATCC 33081, Arthrospira fusiformis UTEX 2721, Arthrospira platensis UTEX 3086, Chlorella vulgaris NREL 4-C12, Chlorella autotrophica CCMP 243, Chlorella sorokiniana DOE1044, Chlorella sorokiniana DOE 1116, Chlorella sorokiniana DOE 1412 (UTEXB3016), Chlorella vulgaris LRB AZ-1201, Chlorococcum littorale UTEX 117, Chlorococcum sp. UTEX-B P7, Chloromonas reticulata CCALA 870, Coelastrella sp. DOE 0202, Cyanobacterium sp. AB1, Micractinium reisseri NREL 14-F2, Microchloropsis gaditana CCMP1894, Microchloropsis salina CCMP 1776, Monoraphidium sp. MONOR1, Monoraphidium minutum 26B-AM, Nannochloropsis oceanica CCAP 849/10, Oscillatoria cf. priestleyi CCMEE 5020.1-1, Picochlorum celeri TG2-WT-CSM/EMRE, Picochlorum oklahomensis CCMP 2329, Picochlorum renovo NREL 39-A8, Picochlorum soloecismus DOE 101, Porphyridium cruentum CCMP 675, Scenedesmus acutus LRB-AP-0401, Scenedesmus obliquus DOE 0152.z, Scenedesmus obliquus UTEX393,Scenedesmus rubescens NREL 46B-D3, Scenedesmus sp. IITRIND2, Stichococcus minor CCMP 819, Stichococcus minutus CCALA 727, Synechococcus elongatus UTEX2973.1, Tetraselmis striata LANL 1001, Tisochrysis lutea CCMP 1324, and Tribonema minus UTEXB3156. For each strain, the identity and the presence of bacterial cohorts was determined using 18S and 16S rDNA sequencing, respectively. The maximum specific growth rate versus temperature data were also used to determine the activation energies (Arrhenius equation) for most strains. For all strains, the measured salinity and temperature tolerance data were compared to those reported in the literature. The fastest growing strains were down-selected for subsequent biomass productivity measurements in climate-simulation photobioreactors, as reported in the next paper in the issue.
18S and 16S rDNA sequencing↗
Examining the Relationship Between the Testate Amoeba Hyalosphenia papilio (Arcellinida, Amoebozoa) and its Associated Intracellular Microalgae Using Molecular and Microscopic Methods
Symbiotic relationships between heterotrophic and phototrophic partners are common in microbial eukaryotes. Among Arcellinida (Amoebozoa) several species are associated with microalgae of the genus Chlorella (Archaeplastida). So far, these symbioses were assumed to be stable and mutualistic, yet details of the interactions are limited. Here, we analyzed 22 single-cell transcriptomes and 36 partially-sequenced genomes of the Arcellinida morphospecies Hyalosphenia papilio, which contains Chlorella algae, to shed light on the amoeba-algae association. By characterizing the genetic diversity of associated Chlorella, we detected two distinct clades that can be linked to host genetic diversity, yet at the same time show a biogeographic signal across sampling sites. Fluorescence and transmission electron microscopy showed the presence of intact algae cells within the amoeba cell. Yet analysis of transcriptome data suggested that the algal nuclei are inactive, implying that instead of a stable, mutualistic relationship, the algae may be temporarily exploited for photosynthetic activity before being digested. Furthermore, differences in gene expression of H. papilio and Hyalosphenia elegans demonstrated increased expression of genes related to oxidative stress. Together, our analyses increase knowledge of this host-symbiont association and reveal 1) higher diversity of associated algae than previously characterized, 2) a transient association between H. papilio and Chlorella with unclear benefits for the algae, 3) algal-induced gene expression changes in the host.
59 BASIC BIOLOGICAL SCIENCES↗
Utilization of microalgae for agricultural runoff remediation and sustainable biofuel production through an integrated biorefinery approach
Generally wastewater such agricultural runoff is considered a nuisance; however, it could be harnessed as a potential source of nutrients like nitrates and phosphates in integrated biorefinery context. In the current study, microalgae Chlorella sp. S5 was used for bioremediation of agricultural runoff and the leftover algal biomass was used as a potential source for production of biofuels in an integrated biorefinery context. The microalgae Chlorella sp. S5 was cultivated on Blue Green (BG 11) medium and a comprehensive optimization of different parameters including phosphates, nitrates, and pH was carried out to acquire maximum algal biomass enriched with high lipids content. Dry biomass was quantified using the solvent extraction technique, while the identification of nitrates and phosphates in agricultural runoff was carried out using commercial kits. The algal extracted lipids (oils) were employed in enzymatic trans-esterification for biodiesel production using whole-cell biomass of Bacillus subtilis Q4 MZ841642. The resultant fatty acid methyl esters (FAMEs) were analyzed using Fourier transform infrared (FTIR) spectroscopy and gas chromatography coupled with mass spectrometry (GC–MS). Subsequently, both the intact algal biomass and its lipid-depleted algal biomass were used for biogas production within a batch anaerobic digestion setup. Interestingly, Chlorella sp. S5 demonstrated a substantial reduction of 95% in nitrate and 91% in phosphate from agricultural runoff. The biodiesel derived from algal biomass exhibited a noteworthy total FAME content of 98.2%, meeting the quality standards set by American Society for Testing and Materials (ASTM) and European union (EU) standards. Furthermore, the biomethane yields obtained from whole biomass and lipid-depleted biomass were 330.34 NmL/g VS added and 364.34 NmL/g VS added , respectively. In conclusion, the findings underscore the potent utility of Chlorella sp. S5 as a multi-faceted resource, proficiently employed in a sequential cascade for treating agricultural runoff, producing biodiesel, and generating biogas within the integrated biorefinery concept.
09 BIOMASS FUELS↗
Mychonastes desiccatus Brown sp. nova (Chlorococcales, Chlorophyta)--an intertidal alga forming achlorophyllous desiccation-resistant cysts
An intertidal Chlorella-like alga Mychonastes desiccatus Brown sp. nova, capable of forming achlorophyllous desiccation-resistant cysts, has been grown in unialgal culture. This small alga was first isolated from a dried sample of a well-studied microbial mat. The mat, located at North Pond, Laguna Figueroa, San Quintin, Baja California, Mexico, is a vertically-stratified microbial community which forms laminated sediments. Morphology, pigment composition and G+C content are within the range typical for the genus Chlorella s. 1. Unlike other chlorellae, however, upon desiccation M. desiccatus forms an achlorophyllous, lipid-filled cyst (thick-walled resting stage) in which no plastid is evident. Rewetting leads to chloroplast differentiation, excystment and recovery of the fully green alga. During desiccation, sporopollenin is deposited within a thickening cell wall. Encystment cannot be induced by growth in the dark. The formation of desiccation-induced cysts allows the alga to survive frequent and intermittent periods of dryness. These chlorellae tolerate wide ranges of acidity and temperature; they both grow and form cysts in media in which sodium ions are replaced with potassium. Although the cysts tolerate crystalline salts, the cell grow optimally in concentrations corresponding from three-quarters to full-strength seawater.
NASA Discipline Exobiology↗
A streamlined approach to characterize microalgae strains for biomass productivity under dynamic climate simulation conditions
Screening microalgae strains under static light and temperature flask conditions cannot directly quantify the biomass productivities of algae growing in the dynamically fluctuating light and temperature conditions of outdoor ponds. In this effort, we describe a testing pipeline that screens for productivity under climate simulated conditions. A validated, miniaturized Laboratory Environmental Algae Pond Simulator (mini-LEAPS) photobioreactor was used to determine optimal medium salinities and biomass productivities of cold-tolerant microalgae collected from Arctic, subarctic, Antarctic, and subalpine habitats. Strains characterized in this effort include: Chlorella antarctica UTEX1959, Chlorella sp. UTEXSNO69, Chloromonas rosae UTEXSNO11, Phaeodactylum tricornutum UTEX646, and Stichococcus minutus CCALA727. Observed productivities of the characterized strains ranged from 1.74 ± 0.25 to 8.18 ± 0.81 g m –2 day –1 (as ash-free dry weight). For each strain, a temperature tolerance profile was generated to identify the most appropriate season (s) for cultivation. The two most promising strains, Chlorella sp. UTEX SNO69 and P. tricornutum UTEX646, were tested alongside the DISCOVR winter benchmark strain Monoraphidium minutum 26B-AM in outdoor open ponds at the PNNL Algae Testbed (PAT) in Arizona. Further, under cold-season outdoor pond conditions, Chlorella sp. UTEXSNO69 achieved average areal biomass productivities of 8.21 ± 0.50 g m –2 day –1 , not significantly different from that of the benchmark strain (8.52 ± 0.43 g m –2 day –1 , p > 0.05). Under spring outdoor pond conditions, P. tricornutum UTEX646 achieved average areal biomass productivities 10.34 ± 0.27 g m –2 day –1 , not significantly different from that of the benchmark (10.07 ± 0.50 g m –2 day –1 ) under the tested conditions (p > 0.05). The streamlined pipeline was thus demonstrated to successfully characterize productive microalgae strains for outdoor deployment by weeding out algae strains that were not productive under the tested dynamic light and temperature conditions.
59 BASIC BIOLOGICAL SCIENCES↗
Photosynthetic Gas Exchange in the Closed Ecosystem for Space. Phase II, Part III. Screening for Thermophilic Algae and Mutation Studies
An algal screening and mutation study was undertaken to obtain algae superior to Chlorella 71105 for use in a photosynthetic gas exchanger. Of the forty-four thermophilic algae studied, eighteen appeared to have growth rates as great as Chlorella 71105. Optimization of the physical and chemical environments of these strains is recommended as a way to further improve growth rates and concomitant oxygen production. The mutation study revealed that Chlorella 71105 is relatively resistant to germicidal ultraviolet radiation. No high temperature mutants of Chlorella 71105 were found.
ALGAE↗
Transport and Retention of Particulate Organic Matter in Sand: Lab Experiments and Modelling
Flow-through reactor (FTR) experiments were conducted at three different downward vertical flow rates to study the transport and retention of particulate organic matter (Chlorella powder) in riverbed sediments. This data package includes data files (.csv) regarding effluent collected from the FTRs during the experiments (time, duration, volume of each sample collection; concentration of nonreactive bromide tracer; concentration of suspended Chlorella determined by absorbance measurements) and sediment (sand) slices collected from the FTRs after the experiments (mass of Chlorella retained in 1 cm depth intervals determined by loss-on-ignition method). More detailed descriptions of data are provided in the data dictionary, and detailed methods are available in the associated MSc thesis listed in ‘Related References.’ The data dictionary and file level meta data (FLMD) are included in the data package as both .csv and .xlsx files. This data package also includes timelapse videos (.mp4) of the experiments, and modelling scripts associated with the experiments. The modelling scripts are included as MATLAB ‘live scripts’ (.mlx, which can be run using MATLAB), and are also included as .pdf and .html files with output plots included (which can be viewed without a MATLAB license and installation). The data files and modelling scripts are organized into a .zip folder for each of the three FTR experiments (three flow rates).
54 ENVIRONMENTAL SCIENCES↗
The microbial community of Ophrydium versatile colonies: endosymbionts, residents, and tenants
Ophrydium versatile is a sessile peritrichous ciliate (Kingdom Protoctista, class Oligohymenophora, order Peritrichida, suborder Sessilina) that forms green, gelatinous colonies. Chlorophyll a and b impart a green color to Ophrydium masses due to 400-500 Chlorella-like endosymbionts in each peritrich. Ophrydium colonies, collected from two bog wetlands (Hawley and Leverett, Massachusetts) were analyzed for their gel inhabitants. Other protists include ciliates, mastigotes, euglenids, chlorophytes, and heliozoa. Routine constituents include from 50-100,000 Nitzschia per ml of gel and at least four other diatom genera (Navicula, Pinnularia, Gyrosigma, Cymbella) that may participate in synthesis of the gel matrix. Among the prokaryotes are filamentous and coccoid cyanobacteria, large rod-shaped bacteria, at least three types of spirochetes and one unidentified Saprospira-like organism. Endosymbiotic methanogenic bacteria, observed using fluorescence microscopy, were present in unidentified hypotrichous ciliates. Animals found inside the gel include rotifers, nematodes, and occasional copepods. The latter were observed in the water reservoir of larger Ophrydium masses. From 30-46% of incident visible radiation could be attenuated by Ophrydium green jelly masses in laboratory observations. Protargol staining was used to visualize the elongate macronuclei and small micronucleus of O. versatile zooids and symbiotic algal nuclei. Electron microscopic analysis of the wall of the Chlorella-like symbiont suggests that although the Ophrydium zooids from British Columbia harbor Chlorella vulgaris, those from Hawley Bog contain Graesiella sp. The growth habit in the photic zone and loose level of individuation of macroscopic Ophrydium masses are interpretable as extant analogs of certain Ediacaran biota: colonial protists in the Vendian fossil record.
Non-NASA Center↗
An algal-bacterial symbiotic system of carbon fixation using formate as a carbon source
The commercial implementation of algal cultures for efficient capture of CO 2 from fossil-derived flue gases is not a reality yet due to several major challenges, particularly low gas-liquid mass transport efficiency along with loss of productivity due to pests. This study has explored an algal-bacterial symbiotic system to utilize formate as a carbon source. The algal-bacterial assemblage, after a 400-day adaptive evolution using the formate medium, has demonstrated a new capability to assimilate both formate and CO 2 to promote biomass production. The formate tolerant culture system also addresses CO 2 mass transfer limitation under high light conditions. Continuous cultivation of the assemblage on formate led to a carbon capture efficiency of 90 % with a biomass concentration of 0.92 g L -1 and biomass productivity of 0.31 g L -1 day -1 , and the system did not require CO 2 aeration. In addition, 13 C isotope tracing, proteomics, and microbial community analysis reveal a uniquely evolved community for formate utilizations and robust biomass synthesis, in which Chlorella sorokiniana is a dominant phototroph companying with highly diverse bacterial subpopulations. In the presence of formate, evidence has shown that Chlorella is able to form mutualism with formate utilizing bacteria in semi-continuous cultures, leading to a highly effective photomixotrophic metabolism. Furthermore, this study demonstrates a new route of using electrochemical-derived formate to support mutualistic algae-bacteria biorefinery while the formate as an alternative carbon source could repel pests for outdoor algal cultivations.
59 BASIC BIOLOGICAL SCIENCES↗
Recycling of Nutrients from Dairy Wastewater by Extremophilic Microalgae with High Ammonia Tolerance
This study explored the possibility of incorporating extremophilic algal cultivation into dairy wastewater treatment by characterizing a unique algal strain. Results showed that extremophilic microalgae Chlorella vulgaris CA1 newly isolated from dairy wastewater tolerated a high level of ammonia nitrogen (2.7 g/L), which was over 20 times the ammonia nitrogen that regular Chlorella sp. could tolerate. The isolate was mixotrophically cultured in dairy effluent treated by anaerobic digestion (AD) for recycling nutrients and polishing the wastewater. The highest biomass content of 13.3 g/L and protein content of 43.4% were achieved in the culture in AD effluent. Up to 96% of the total nitrogen and 79% of the total phosphorus were removed from the dairy AD effluent. The ability of the algae to tolerate a high level of ammonia nitrogen suggests the potential for direct nutrient recycling from dairy wastewater while producing algal biomass and high value bioproducts.
54 ENVIRONMENTAL SCIENCES↗
Preliminary development and evaluation of an algae-based air regeneration system
The potential of air regeneration system based on the growth of microalgae on the surface of porous ceramic tubes is evaluated. The algae have been maintained in the system for extended periods, up to 360 days. Preliminary measurements of the photosynthetic capacity have been made for Chlorella vulgaris (UTEX 259), Neospongiococcum punctatum (UTEX 786), Stichococcus sp., and Gloeocapsa sp. Under standard test conditions (photosynthetic photon flux approximately 66 micromoles m-2 s-1, initial CO2 concentration approximately 450 micromoles mol-1), mature tubes remove up to 0.2 micromoles of CO2 per tube per minute. The rate of removal increases with photon flux up to at least 225 micromoles m-2 s-1 (PPF); peak rates of 0.35 micromoles of CO2 per tube per minute have been achieved with Chlorella vulgaris. These rates correspond to between 120 and 210 micromoles of CO2 removed per square meter of projected area per minute.
Non-NASA Center↗
Nitrogen derived from Combined Algal Processing supports algae cultivation for biofuels
Algae biomass, a biofuel feedstock with potential to reduce global CO 2 emissions, is currently too expensive Nitrogen, a significant input and life cycle cost, requires research on recycling and additional sources to reduce costs. Integral to nitrogen recycling is the knowledge of actual nitrogen requirements in outdoor ponds, thus data from the Unified Field Studies of the Algae Testbed Public-Private Partnership was analyzed. The data showed that Nannochloropsis oceanica used 14–24 mg/L NH 4 + per harvest cycle for robust growth. Nitrogen recycled directly from the algae conversion process will likely be the primary source of nitrogen for algae cultivation. Harvested algae can be fractionated via Combined Algal Processing (CAP), where algae are pretreated, fermented, and the fuel feedstocks (e. g. ethanol, lipids) are removed leaving a nitrogen rich slurry. Chlorella vulgaris grew robustly in nitrogen-free media supplemented with CAP residues through 4 harvest cycles with no inhibition. The amount of nitrogen as free ammonium available for recycling directly without further processing was insufficient (~4% of total needed), thus the nitrogen fixing bacteria, Azotobacter vinelandii, was investigated as a means for increasing ammonium concentration in CAP residues. This organism was shown to be proficient at growth on sugars liberated from algae biomass during pretreatment and sustained algae growth using A. vinelandii supernatants was also demonstrated. Using Azotobacter to increase ammonium concentrations on-site using an inexpensive biological process to generate the necessary ammonium for outdoor algae cultivation improves sustainability of algae biofuels by avoiding the energy intensive steps of typical ammonium production and transport. Furthermore, these results show that byproducts of algae conversion begin to close nitrogen demand and, with further supplementation by diazotrophic bacteria or protein hydrolysis, may completely replace current energyintensive and costly industrial nitrogen sources offering a path to increased economic feasibility and improved life cycle analysis of algae-based biofuels.
09 BIOMASS FUELS↗
Reused cultivation water from a self-inhibiting alga does not inhibit other algae but alters their microbiomes
Economical production of algal commodities (food, feed, and fuels) requires reusing cultivation water to reduce operating costs. While some algae strains show growth inhibition in reused water, other strains appear un-affected. Reusing water to grow different strains (i.e., crop rotation) could potentially improve overall biomass production compared to water reuse with the same strain by reducing accumulation of strain-specific waste products, limiting carryover of population-specific pathogens such as viruses, and introducing bacteria that degrade residual organic matter. Here, batch culture experiments tested the effects of reused cultivation water from the self-inhibiting diatom Staurosira sp. C323 on the diatom Navicula sp. SFP and green alga Chlorella sp. D046, as well as their microbiomes. Navicula sp. and Chlorella sp. grew well in Staurosira sp. reused water, indicating that previously observed self-inhibition was likely specific to this Staurosira strain. While algal mi-crobiomes were not significantly different between controls and reused water treatments, specific bacteria taxa were differentially abundant in reused treatments, suggesting that some taxa responded to compounds re-maining in the reused water. Finally, results suggest that algae growth responses in reused water may depend largely on strain-specific factors, and that crop rotation can support biomass production.
09 BIOMASS FUELS↗
A validated thermal and biological model for predicting algal productivity in large scale outdoor cultivation systems
Proper assessment of the sustainability of algal products requires an understanding of algal growth rate with regional and temporal resolution. This study developed a bulk growth model that utilizes geospatial data, reactor geometry inputs, and a maximum of six strain-specific parameters to predict algal productivity with temporal and regional resolution. The model requires local weather data to calculate the time-resolved culture temperature which is combined with a biological model based on maximum theoretical carbon fixation to predict the time-resolved algal concentration. Validation of the model against experimental data illustrates an average ac- curacy of 0.27% ± 5.32% when comparing algal concentration at harvest over 45 days of cultivation data for Galdieria sulphuraria 5587.1, and Galdieria sulphuraria Soos grown in photobioreactors (PBRs) in Mesa, AZ (33.4152° N, 111.8315° W). For open raceway ponds (ORPs), an accuracy of 0.9% ± 2.35% was achieved when comparing algal concentration at harvest over 50 days of cultivation data for Chlorella vulgaris, Desmodesmus intermedius, Nannochloropsis oceanica grown in Atlanta, GA (33.7490° N, 84.3880° W) and Mesa, AZ (33.4152° N, 111.8315° W). The validation trials spanned three seasons (winter, spring, and summer) to ensure model ac- curacy throughout the year, and concentration between harvests was modeled with hourly resolution. The validated model is used to assess the productivity at several case-study locations in the United States with Chlorella vulgaris, grown in open raceway ponds and Galdieria sulphuraria grown in vertical flat panel photo- bioreactors. In conclusion, the model has been published as an open-source tool, in an effort to increase the fidelity of future studies that rely on outdoor algae cultivation for feedstock production and allow for time resolved results for future techno-economic and life cycle assessments.
59 BASIC BIOLOGICAL SCIENCES↗
DISCOVR strain screening pipeline – Part III: Strain evaluation in outdoor raceway ponds
Not Available
59 BASIC BIOLOGICAL SCIENCES↗