Patent Application
20150299646
Despite significant investment in research and development, commercial viability of algal-derived biofuels remains a future prospect. Costs of mass algal culture, including commercially available nutrient stocks such as f/2 media cost $25/liter unpredictability of algal stocks (see Ravi, et al. 2013), high costs of algal concentration and extraction of products and limited choices for algal stocks all contribute to the untenable costs of algal biofuel—in excess of $17 per gallon—and the limited use of this energy source in the open market (Ravi, et al., 2013).
Sewage sludge is rich in various nutrients. Analyses of sewage sludge samples from 6 north-central states in USA yielded median concentrations as follows: N, 4.2; P, 3.0; K, 0.3%; Pb, 540; Zn, 1,890; Cu, 1,000; Ni, 85; and Cd, 16 mg/kg, and for aerobically treated sludges: N, 4.8; P, 2.7; K, 0.4%; Pb, 300; Zn, 1,800; Cu, 970; Ni, 31; and Cd, 16 mg/kg (Sommers 1977). Sewage is a source of nutrients both organic and inorganic that sustain algal growth. Growth of these algae could result in blooms either benign or toxigenic which could have serious environmental and societal impacts.
Microalgae synthesize organic material from inorganic material via photosynthesis which can be expressed as: 6CO2+6H2O+light 8 photons C61412O6+6O2↑
During photosynthesis, microalgae assimilate macronutrients (N, P, S) and the trace elements (Fe, Zn, Mn) as expressed below:
106CO2+16NO3+PO4+SO4+10−2Fe+4×10−3Zn+4×10−4Mn(C106H263O110N16PS)+138O2↑
Organic matter (C106 H263 O110 N16 PS) and oxygen are the two most important products.
Traditionally microalgal cultures both marine and freshwater are grown in media with high concentrations of nutrients (Table 1A) that are several orders of magnitude higher than those in the marine environment (Table 2A):
0.1 μm-17.6 μM
1.0 μM-1.8 mM
Cultures of microalgae have the potential for bioremediation because of their ability to assimilate and bioaccumulate several nutrients. Under defined culture conditions of temperature (25-27° C.) and fluorescent light with a light:dark photoperiod of 15 h:9 h, the microalgae Tetraselmis chuii and Nannochlopropsis sp. have been utilized for removal of nutrients in recirculation aquaculture systems in waste water (Sirakov and Velichikova 2014). N. oculata removed 78.4% of total nitrogen, 92% of nitrate and 42.3% phosphate. Utilizing bacterial-biofilm bioreactors higher rates of removal i.e. 91±3%, 70±8% and 85±9% for carbon, nitrogen and phosphorus, respectively, are also possible (Posadas et al. 2013). Chlorella vulgaris and algae taken from Pleasant Hill Lake, Ohio grown under defined conditions were used for bioremediation of wastewater laden with nitrogen, phosphorous, chromium (Cr (VI)) and cadmium (Cd (II) (Saikumar 2014).
Most of the microalagal cultures are raised under defined conditions of temperature and under a bank of growlux fluorescent lights which escalate production costs (Table 3A). The key to successful bioremediation would be to raise microalgal cultures in waste water under ambient conditions of light and temperature. Incidentally this would remove the nutrients from the wastewater via bioaccumulation by microalgae.
Pavlova lutheri,
Nannochloropsis sp.
Tetraselmis suecica
Nannochloropsis sp
Tetraselmis suecica
Cyclotella cryptica
Nitzschia alba
Chlorella sp.
Cyclotella
Chlorella sp.
Crypthecodium cohnii
Schizochytrium sp
Provided herein is an isolated and purified new microalgal species designated Scenedesmus species Novo and progeny thereof. The alga was collected at latitude 35.769 and longitude 106.692. It is capable in culture including TAP medium of producing a biomass of about 10.41×106 cells per ml and at least about 4 μg per ml, for example, about 4.18 to about 4.5 μg per ml, of carotene under outdoor growth conditions comprising temperatures reaching 40° C. or higher.
The new microalgal species has an 18S ribosomal RNA gene sequence [SEQ ID NO:1] at least about 99% to about 100% identical to SEQ ID NO:1, and about 98% identical to algal species G24 (38).
In embodiments, the alga is capable of producing up to at least about 3.58 pg per cell of carotenes under indoor growth conditions. The term “up to at least about” as used with respect to a numerical value herein refers to a value seen at any point on a graph of such values over time.
The cultures can be cultivated in sewage/wastewater at ambient temperatures of up to at least about 40° C. In embodiments the cultures are cultivated at temperatures above 40° C., for example between about 40° C. and about 100° C., or between about 40° C. and about 80° C., or between about 40° C. and about 60° C., or between about 40° C. and about 50° C. As used herein, the term “extremophilic microalgae” refers to thermophilic microalgae capable of growth at such temperatures. The microalga of the present invention may be used to treat sewage/wastewater (it is a freshwater microalga) and provides high production of hydrocarbons, especially carotenoids and provides bioremediation of the sewage/wastewater making the treated sewage/wastewater far easier to further process in water treatment plants to clean water.
Cultivated in water enriched with growth-promoting nutrients such as those of TAP medium, at ambient room temperatures (e.g., about 20° C. to about 26° C.), cultures of this microalga are capable of producing an average lipid content of between about 63 pg per cell and about 95 pg per cell. Cultures grown in enriched TAP medium indoors can have a chlorophyll α content up to between about 20 and about 49 μg per ml, and a carotene content up to about 10 to about 24 or about 25 μg per ml.
In embodiments, grown outdoors in wastewater at temperatures that reach 40° C. or higher, in a TAP medium, such cultures can have a lipid content of between about 16.7 and 81.4 pg per cell, a chlorophyll α content up to about 5.8 μg ml−1, and a carotene content over 4 μg ml−1, e.g., about 4.18 to about 4.5 μg ml−1.
The cultured algae are circular and can be single cells and/or clumps of up to about 360 cells which drop to the bottom of the vessel containing the culture, thus making it easy to harvest the cells. Harvested algal biomass produced by the microalgae can be dried to a mass having a water content less than about 5%.
A method for culturing and harvesting extremophilic microalgae is also provided herein. The method comprises preparing a growth medium composition comprising said extremophilic microalgae and water (including sewage/wastewater) comprising nutrients capable of enhancing growth of the microalgae; allowing the microalgae to proliferate in the composition under ambient outdoor conditions comprising intervals of ambient temperatures of at least about 40° C. and ambient light of up to about 1400 to about 1600 watts; and dewatering the composition and recovering and drying it to obtain an algal biomass comprising the microalgae and less than about 5% water content. This same method or a similar method may be readily adapted for use on sewage/municipal wastewater for bioremediation of the sewage/wastewater, making it far more easy to process in water treatment plants.
The dewatering step can be performed in a micro solid-liquid separation system such as one from AlgaeVenture Systems, Marysville, Ohio. In preferred embodiments, the extremophilic microalgae in the growth composition are Scenedesmus species Novo. In embodiments, the growth composition also comprises wastewater, often municipal wastewater (sewage). In embodiments, the nutrients in the growth composition are selected from the group consisting of TAP medium components, selenium, boron and iron. The wastewater can be sterilized urban or agricultural wastewater or nonsterilized urban or agricultural wastewater. The wastewater may also be industrial or residential wastewater, often residential wastewater or a combination of residential (municipal) wastewater and industrial wastewater. Any wastewater in which the microalgae of the present invention may grow represents a source of nutrients which be converted by the microalgae of the present invention. Thus, the present invention may be used to convert sewage/wastewater to useful lipids and hydrocarbons, especially including carotenes in high concentrations under conditions in which most microalgae are incapable because of the extreme conditions of certain embodiments of the present invention and to bioremediate the sewage/wastewater to make it less dangerous and more easy to process to clean water (e.g. in water treatment plants).
In another embodiment hereof, a method for culturing and harvesting extremophilic microalgae is provided comprising: preparing a growth composition comprising the extremophilic microalgae and water, which often constitutes sewage/municipal wastewater and often further comprises TAP medium components in amounts sufficient to enhance growth of said microalgae; allowing the microalgae to proliferate in said composition at room temperatures, such as temperatures of about 23° C. to about 25° C., or higher; and dewatering and drying the composition and recovering an algal biomass comprising the microalgae and less than about 5% water content. In embodiments of this method, the microalgae are Scenedesmus species Novo. In embodiments, the growth composition comprises sewage/wastewater and further includes the components of TAP medium and optionally further components such as selenium, boron and iron, among others.
A method of inhibiting growth of a microorganism is also provided herein. The method comprises contacting cells of the microorganism with an extract of Scenedesmus species Novo. The microorganisms can be bacteria, viruses, parasites, or fungi.
Applicants have isolated an extremophile green alga, Scenedesmus species Novo, with unique growth and biochemical characteristics, from Jemez warm water springs in New Mexico. Sequencing 18S rDNA confirmed the alga as a new species. Cultures in TAP medium at 24±1° C. at continuous light (132-148 μmol photons m−2s−1) attained peak biomass levels of 27.4×106 cells ml−1 with a division rate (k) of 0.54 day−1, and yielded 49.11 μg chlorophyll α ml−1 and 24.93 μg carotene ml−1 on the 7th day high levels of biomass were sustained in sterilized or unsterilized municipal wastewater, either enriched with 1% TAP nutrients or unenriched. Under outdoor conditions (6524-7360 μmol photons m−2s−1 and ˜40° C.), high levels of biomass (10.41×106 cells ml−1), and yields of 8.92 μg chlorophyll α ml−1, and 4.18 μg carotene ml−1 were sustained. Lipids in cells raised in TAP under controlled, less severe conditions ranged from 63 to 94.3 pg cell−1, and in outdoor wastewater 16.7 to 81.4 pg cell−1, which are higher than those previously reported in the literature. In cultures raised in TAP in outdoor waste water, lipid (% of cell dry weight) ranged from about 15% to about 74%, substantially higher than previous literature values. Total carotenoids ranged between 0.37 and 3.58 pg cell−1. Thus, in preferred embodiments, the microalgae may be used in freshwater, making it particularly useful to treat sewage/municipal wastewater to produce high concentrations of hydrocarbons, especially carotenes, and can be used to make the sewage/wastewater far less polluted and more amenable and easier to process to clean water. Moreover, the microalgae may be used at varying temperatures from room temperature to temperatures of up to at least about 40° C. to about 100° C. or more (depending on the pressure of the medium in which the microalgae is grown).
Because of its ability to produce high levels of microalgal biomass in wastewater under harsh ambient climatic conditions and yield of high levels of lipids and carotenes, mass cultivation of Scenedesmus species Novo is useful in many biotechnological applications. Because of the extremophilic nature of Scenedesmus species Novo, this microalgae is particularly suited for industrial use because it can tolerate high temperatures which often will cause difficultires for other microalgae and microorganisms in culture. Accordingly, the microalgae of the present invention, because of its extremophilic stability and its ability to grow in fresh water culture (making it useful for sewage/wastewater treatment compared to salt water species) and produce high concentrations of lipids and/or carotenoids in culture, providing methods of the present invention that are more reliable, resilient and cost effective than prior art approaches.
The following terms are used throughout the specification to describe the present invention. Where a term is not given a specific definition herein, that term is to be given the same meaning as understood by those of ordinary skill in the art. The definitions given to the disease states or conditions which may be treated using one or more of the compounds according to the present invention are those which are generally known in the art.
The singular forms “a,” “an,” and “the,” include plural referents unless expressly and unequivocally limited to one referent. Thus, for example, reference to “an inhibitor” can include two or more different compounds. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or other items that can be added to the listed items.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, 2001, “Molecular Cloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols in Molecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: A Laboratory Handbook” Volumes I-III; Coligan, ed., 1994, “Current Protocols in Immunology” Volumes I-III; Gait ed., 1984, “Oligonucleotide Synthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”; Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney, ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized Cells And Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”
“Wastewater” includes, but is not limited to, contaminated surface and subsurface runoff water from storm events and acid mine drainage, coking wastewater generated in the high-temperature carbonization of raw coal, coal gas purification and refining process of chemical products, raw untreated sewage wastewater having a significant concentration of waste solids, water comprising any number of pollutants found in urban, residential and agricultural settings around the world, storm runoff which picks up a wide variety of contaminants as it flows across the surface and then into private and public waters, runoff that flows across roads and parking lots and that picks up oil, grease and metals from automobile discharges, or that picks up nitrate and phosphate from fertilized lawns and golf courses, or that picks up organic waste, herbicides and pesticides from agricultural sites, or that picks up grit and colloidal particles from all of these locations, water sources impacted by mining, which include surface and subsurface flows, water containing a wide variety of pollutants related to hydrologic fracturing for natural gas as well as acidified mine drainage water carrying heavy loads of dissolved metals and waters such as streams, rivers and lakes, aquifers and groundwater containing any contaminant. In certain preferred embodiments according to the present invention, the wastewaster used is urban wastewater, often industrial or municipal/domestic wastewater or a combination of municipal/domestic wastewater and industrial wastewater (from standard sewage runoff). The use of sewage/municipal wastewater is preferred. Domestic wastewater includes wastewater from residential settlements and services which originates predominantly from the human metabolism and from household activities.
The term “microalgae” refers collectively to unicellular organisms that have photosynthetic pigments and are photosynthesized. Microalgae can grow in the presence of a suitable amount of light and dissolved nutrients and can be utilized in various applications, including the production of biomass and biofuel and the improvement of atmospheric and aquatic environments. The preferred microalgae for use in the present invention is Scenedesmus species Novo.
Microalgae have several advantages as feedstock to land-based biofuels. They are renewable and amenable for mass cultivation on nonarable land; they can be a source of significant quantities of lipids; they act as a source of value-added co-products; they can be used for bioremediation; and they are capable of sequestering carbon. Microalgal biomass can yield between 58,700 and 90,000 liters of biodiesel per hectare per year (1,2,3). Biofuels contribute to ˜2% of global transport fuel today but are predicted to increase to 27% by the year 2050 (4). For biotechnological applications, sustenance and steady supply of algal biomass are required, which is feasible by mass cultivation of algae. Only a small percentage of the 17,500 microalga species are cultured and about 50 have been screened for their utility—mostly in biofeed, with only a few having been identified as useful for biofuel. Most of the algal isolates are from temperate waters and are grown in defined sterile media under controlled conditions of temperature and light, which collectively escalate biomass production costs to as high as $7.32 per kg of algal biomass and $24.60 per liter algal oil (5).
Microalgae characterized as extremophiles remain least studied. Extremophile algae can readily adapt to exacting local physicochemical conditions, and manifest biochemical and physiological responses such as the production of carotenoids, as in Dunaliella salina (6). The extremophile diatom Nitzschia frustula and the green alga Chlamydomonas plethora isolated from the semiarid harsh climate of the Arabian Gulf (7) have high division rates, carbon assimilation rates (18.1 22.8 to mg C per mg chlorophyll α per hour) approaching their theoretical maxima as well as yielding levels of acids and leucine, lysine, glutamic acid and arginine that make them valuable in biotechnological applications.
Reported here are observations on Scenedesmus species Novo, an extremophile green alga isolated by us from Jemez Springs, N. Mex. This alga grows well in urban wastewater under ambient conditions of light and temperature in New Mexico, yields considerable quantities of lipids and carotenoids and is especially useful for producing algal biofuel.
The invention is illustrated further in the following non-limiting examples.
Several samples of water were collected from Jemez warm water springs (latitude 35.769 and longitude 106.692) and enriched with nutrients f/50, f/10 (28) and TAP media. Samples were incubated at 24±1° C. at continuous 132-148 μmol m−2s−1 light supplied by cool white fluorescent lights. Using repeated serial dilution techniques algal cultures were established. Pure cultures were based on isolates established by streaking on agar plates. Agar slants were based on enrichments with f/50, BG11 and TAP media. Utilizing usual sterile culture techniques, colonies were isolated and gradually scaled up into BG11 (29) and modified TAP medium (30). TAP medium based on enrichment with 10 ml each of triacetate stock, nutrient stock, phosphate buffer and trace elements supported excellent growth. Trace element enrichment follows the formula as described in Hunter (1950) (31).
Cultured cells of Scenedesmus species Novo were circular, either singular or in clumps up to 356 cells and did not have any spines. Cells were non-motile, enveloped in mucilage (
All algae samples collected from this location exhibited similar properties and were considered to be samples of the same species.
All growth experiments were done in triplicate. Samples were incubated at 24±1° C. at continuous 132-148 μmol m−2s−1 light supplied by fluorescent lights, or were incubated over the terrace of a building under natural light (1400-1600 watts m-2, equivalent to 6524-7360 μmol m−2s−1), and ˜40° C. Suitable aliquots were drawn from each culture aseptically for enumeration, chlorophyll a and carotenoid determinations. Direct counts were made on the samples using an Improved Neubauer haemocytometer.
Based on direct cell counts generative times in hours were calculated (33). The division rate of cells was 0.54 day−1 in TAP medium and 0.27 day−1 in BG 11 (Table 1).
For chlorophyll α and carotenoids, a one-ml sample was centrifuged into a pellet and sonicated with a Branson sonicator with a fine probe for one minute at 0° C. in ice cold 90% acetone. The contents were thoroughly mixed in a vortex mixer and extracted for 24 h at 4° C. in a refrigerator sufficient for complete extraction. The extracts were cleared by centrifugation in a Beckman CS 15R centrifuge, and their absorptions at 750 (blank), 664, 647, and 452 nm, were read in a Spectromax spectrofluorimeter that accommodates 96 well polypropylene NUNC plates.
The following equations were used to calculate pigment concentrations (μg ml−1 culture):
Chl α=11.93D664−1.93D647(Vc/Vs) (34)
Carotenoid=3.86*D452(Vc/Vs) (35)
where Vc=volume of culture sample (ml) and Vs=Volume of extract (ml).
Quantitative measurement of fatty acids was performed by Avanti Polar Lipids, Inc. (www.Avantilipids.com) of fatty acid methyl ester (FAME) by gas chromatography with flame ionization (GC/FID) on 1.5 ml of extracted algae using 7-level calibration curves of FAME standards for C8-C24:1 compounds with a C15:1 as internal standard (36). Each sample was injected in triplicate. Standard deviation of the mean ranged between 0.01 and 0.04 when the mean total lipids were <6.0, and between 0.71 and 2.71 when the means were 15.35 to 22.64.
Two-way analysis of variance (ANOVA) was done on several variables using an EXCEL statistical package (37) to test significance of differences between treatments.
Wastewater Media
Filtered Albuquerque wastewater was enriched with TAP stock solutions nutrients (one ml each to 0.2 μm filtered liter of waste-water) and used either sterilized or unsterilized depending on the experimental design. The media were designated as: ST—Sterile Wastewater enriched with 1% TAP; NST—Non-sterile Wastewater enriched with 1% TAP; WWS—Sterile Wastewater; WWNS—Nonsterile wastewater.
Algal cells grew readily in TAP medium and reached peak biomass levels (27.4×106 cells ml−1 (
Cultures raised in the laboratory at 24±1° C. at continuous 132-148 μmol m−2s−1 light in sterile wastewater enriched with 1% TAP supported good growth and yielded 10×106 cells ml−1, 17.6 μg chlorophyll α ml−1, and 7.42 μg carotene ml−1 (Table 1). The division rate was 0.24 day−1 (Table 1). Growth in non-sterile wastewater, although enriched with 1% TAP, was 5.39×106 cells ml−1, yielding 6.79 μg chlorophyll α ml−1, and 3.69 μg carotene ml−1. However growth was high in unenriched sterile wastewater 10.18×106 cells ml−1, yielding 12.08 μg chlorophyll α ml−1, and 7.64 μg carotene ml−1 (Table 1), higher than in unenriched, nonsterile wastewater that has 4.33×106 cells ml−1, and yields 11.04 μg chlorophyll α ml−1 and 5.56 μg carotene ml−1 (Table 1).
Indoor wastewater cultures had more pigments per cell (range of 2.88 pg cell−1 chlorophyll α to 3.43 pg cell−1 chlorophyll α and 1.52 pg cell−1 to 1.75 pg cell−1 carotene compared to those grown either outdoors or in TAP or BG11 media (Table 1).
Growth of cultures raised on the terrace of a building under harsh ambient conditions of light (1400-1600 watts) and temperature (˜40° C.) favorably compared to that of cultures raised indoors. The cultures raised under these harsh ambient conditions produced a biomass yielding 10.41×106 cells ml−1, 8.92 μg ml−1 chlorophyll α, and 4.18 μg ml−1 carotene (Table 1) in sterile wastewater enriched with 1% TAP; with a division rate of 0.24 day−1. In unenriched sterile wastewater peak biomass was 8.81×106 cells ml−1, yielding 5.82 μgml−1 chlorophyll α and 4.49 ml−1 carotene (Table 1) with a cell division rate of 0.19 day−1. Corresponding numbers for unenriched nonsterile wastewater cultures were 5.08×106 cells ml−1 biomass, yielding 5.41 μgml−1 chlorophyll α, and 3.02 μgml−1 carotene with a division rate of 0.14 day−1 (Table 1).
Results of two-way analysis of variance (Table 2) showed that statistically significant differences existed in the biomass levels depending on the medium utilized. For example cultures grown in the defined TAP medium yielded higher levels of cells, biomass, chlorophyll α, and carotene cell−1, than those in BG11 medium. Cultures grown in sterilized wastewater enriched with 1% TAP nutrients had significantly higher cell densities, chlorophyll α and carotene than those in similar media but unsterilized.
Production of biomass, i.e., cells, chlorophyll α and carotene in cultures grown indoors and outdoors in ST (Sterile medium enriched with 1% TAP), was significantly higher than in cultures grown in NST medium (non-sterile medium enriched with 1% TAP), WWS (sterile wastewater) and WWNS (nonsterile wastewater). However differences in chlorophyll α levels in cultures raised in non-sterile wastewater enriched with 1% TAP (NST) and in non-sterile wastewater (WWNS) were not statistically significant.
A feature of interest is the high initial levels of cellular chlorophyll α, and carotene and their gradual decrease with time (
In outdoor cultures the initial cellular chlorophyll α levels (
Our results show that microalgal extremophiles native to New Mexico can be brought into wastewater culture. Scenedesmus species Novo studied here is especially suited for mass cultivation and for utility in biotechnology. This alga is cultivable in wastewater and under the harsh ambient light and temperature conditions of semiarid regions such as Albuquerque, N. Mex. Its production is cost-effective, an important consideration in biotechnology applications. Biomass levels of our outdoor cultures were high (10.41×106 cells ml−1, yielding 8.92 μg chlorophyll α ml−1, and 4.18 μg carotene ml−1), and division rates (8) compared well with those obtained on cultures raised under measurable controlled, less severe conditions of temperature and light. Harvesting the algal biomass is also simple and cost-effective as our cultured cells settle readily to the bottom and separation does not require centrifugation, flocculation, or utilization of other energy-intensive methods.
A few investigators have studied the lipid as percent dry weight of cultured algae (Table 4); our algal cells had a range of 15-85% (Table 3, Table 4) compared 0.1 to 75% reported on several species (Table 4). Several studies reported potential for sustaining algal blooms in water enriched with wastewater from municipal sewage, agriculture and industrial sources and total lipids that varied between 9 and 29% of dry weight (9). Total lipids in Chlamydomonas reinhardtii were 25.25% dry weight (10); 17.85% in Botryococcus braunii (11), 9-13.6% in Chlorella ponds enriched with dairy manure (12), and 14% to 29% in mixed algae cultures originally isolated from local wastewater treatment ponds (13). Because of their high-value for biofuel, nutraceuticals and pharmaceuticals, carotenoids and lipids from microalgae have been studied, with most investigators reporting these values as percent of cell dry weight, lipid production as mgl−1d−1, gl−1d−1, and g m−2d−1 (1, 14, 9, 15, 16, 17, 18). Preliminary analyses of lipids on our algal slurries (Table 3) showed that lipid yield was initially high, reaching a peak (94.3 pg cell−1) following 8 days of growth.
Cellular carotene in our algal cells ranged from 0.95 to 3.58 pg cell−1 and compared favorably with carotene yields (Table 5) for Dunaliella salina (19) or D. salina, D. bardawil and 18 strains of microalgae isolated from tropical waters of the Bay of Bengal (20).
We have successfully brought the extremophile alga Scenedesmus species Novo, native to New Mexico, into culture.
Sequencing of the new Jemez alga was completed utilizing three different primers to completely sequence the 18S rDNA (32) and the data were used to assemble the contig. The 18S rDNA sequence of Scenedesmus species Novo [SEQ ID NO:1] is shown in the Sequence Listing at the end of this Specification.
Sequencing of the Jemez alga showed that it is most closely related to G24 (but less than 99% homologous to G4, and more distant from Scenedesmus abundans and S. communis.
In the defined TAP medium, under controlled conditions of temperature and light, high levels of biomass (cells), chlorophyll α and carotene and division rates were sustained. Further this extremophile alga grew well in wastewater under controlled conditions of temperature and light and under harsh ambient temperatures and light as well. An added advantage of our cultures is the settlement of cells readily to the bottom which makes their harvesting simple, and cost effective.
Cellular lipids in our cultures are the highest reported for microalgae. Lipids in cultures attained their peak (94.3 pg cell−1) in a relatively short time, remained high and contributed between 57% and 85% of cell weight. Carotenoids were also high (0.95-3.58 pg cell−1) and compared favorably with those obtained on 18 strains of microalgae isolated from the tropical waters of the Bay of Bengal.
Scenedesmus species Novo grows rapidly under harsh climatic conditions and in wastewater. Through biochemical manipulation lipid and carotene synthesis can be regulated in algae. This involves imposing a physiological stress such as nutrient starvation to channel metabolic processes towards accumulation of bioactive compounds. To enhance yield of microalgal biomass, micronutrients such as selenium, boron and iron can be optimized, along with temperature and light.
Antimicrobial Activity of Extracts of Scenedesmus novo
S. novo cells are grown at 22° C. under continuous light conditions for 10 days to achieve a dense culture. Final volume of culture is 2.0 liters. Cells are centrifuged and cell pellets are subjected to lysis using sonication in the setting of proteinase K and bath temperatures of 4° C. to avoid inactivation of proteins. Cell lysate is decanted and tested in antimicrobial screening assays against a control extract prepared from a Chlorella species.
Antimicrobial assays are conducted using turbidity assessments for Minimum Inhibitory Concentrations. Target species of bacteria include E. coli, S. aureus, K pneumonia and P. vulgaris. In all cases, cell lysates of S. novo exhibit inhibition of growth of bacteria at 12 hours in a 96-well plate assay.
Antiparasite assays are conducted as above using 2 target organisms: Trypanosoma cruzi strain “Y” and Leishmania donovani. Cell lysates of S. novo inhibit parasite growth at 24 hours.
Antifungal assays are conducted with Candida albicans and inhibition of fungal growth in a broth assay is observed at 24 hours with cell lysates of S. novo.
Novo in Wastewater enriched with TAP nutrients.
Scenedesmus sp Novo
Scenedesmus sp obliquus
S. obliquus
S. obliquus
Chlorella vulgaris
Chlorella sps.
Chlorella prothecoides
Dunaliella tertiolecta
Neochloris oleabundans
N. oleabundans
Botryococcus braunii
Botryococcus braunii
0.05-0.678.
D. salina
D. bardawil
D. tertiolecta
D. parva
Nannochloropsis sp.
Isochrysis sp.
Tetraselmis sp.
Rhodomonas sp.
Nannochloropsis sp
Scenedesmus
Dunaliella salina
Nannochloropsis
galitana
Haematococcus
pluvialis
Dunaliella
bardawil
D. salina
D. salina
We enriched municipal waste water with 1% TAP (Gorman and Levine 1965) nutrients. This is probably the most widely-used medium at present for experimental work. The following stock solutions were used:
To make the final medium, mix the following:
We have isolated an extremophile green alga Scenedesmus, from Soda Dam warm water springs, New Mexico. Whether grown in water enriched with 1% TAP nutrients or un-enriched, high levels of biomass could be sustained in sterilized or un-sterilized municipal wastewater. Under outdoor conditions (6524-7360 μmol photons m−2 s−1 and ˜40° C.) high levels of biomass (10.41×106 cells ml−1, 8.92 μg chl a ml−1, and 4.18 μg carotene ml−1) could be sustained. Under controlled conditions lipids in cells raised in TAP ranged from 63 to 94.3 pg cell−1 and in outdoor wastewater 16.7 to 81.4 pg cell−1 which are higher than those reported. In cultures raised in TAP medium lipid (% of cell dry weight) ranged from 57 to 85% compared to 15-74% in outdoor waste water which are also substantially higher than literature values. Total carotenoids ranged between 0.37 and 3.58 pg cell−1 compared to 0.24-4.75 pg cell−1 in literature.
Because of its amenability to produce high levels of microalgal biomass in wastewater under harsh ambient climatic conditions, and yield of high levels of lipids and carotenes, Scenedesmus species Novo has the potential to sustain biotechnological applications. Notably, the microalgae biomass can produce biodiesel (Christi 2007), bioethanol (Harun et al. 2010), biogas, and biohydrogen (Demirbas, 2010). and bio-oils. Since the novel alga can be cultured in wastewater, it has potential for bioremediation and production of valuable products. We recommend more isolations of several extremophile algal species native to New Mexico with a view to develop strategies for a viable bio-economy based on their mass cultivation.
All publications referred to herein are incorporated herein by reference to the extent not inconsistent herewith.
Numerical ranges mentioned herein specifically include all numbers to two decimal places that fall between the stated end points of the ranges.
It will be understood that although specific organisms, reagents, method steps and process conditions have been provided herein, equivalents of these are considered to be within the scope of the appended claims.
This application is a continuation-in-part application claiming priority to U.S. patent application Ser. No. 13/723,687 filed Dec. 21, 2012, of identical title, and U.S. Provisional Patent Application Ser. No. 61/579,120 filed Dec. 22, 2011, each of which is incorporated herein by reference in its entirety.
This invention was made at least in part with Government support from the Department of Veterans Affairs. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61579120 | Dec 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13723687 | Dec 2012 | US |
| Child | 14698481 | US |
