Apparatuses, Systems, and Methods for Growing Algae Biomass

Cross-Reference to Related Applications

[0001] This application claims priority to and the benefit of co-pending U.S. provisional patent application Serial No. 62/311,716, entitled System and Method for Growing Algae Biomass, filed March 22, 2016, which is incorporated herein by reference in its entirety.

Statement Regarding Federally Sponsored Research or Development

[0002] Not applicable

1. TECHNICAL FIELD

[0003] The present invention relates to apparatuses, systems, and methods for cultivating algae biomass. The invention also relates to apparatuses, systems and methods for growing auto- flocculating (self-aggregated) species of algae in controlled environments.

2. BACKGROUND

[0004] The global demand for aquafeed is increasing at a compounded annual growth rate of about 11.4% and expected to reach ~ $30 billion by 2020 (Wood, 2014; Smith, 2016). This fact asserts the importance of maintaining a viable supply of feed ingredients. Two protein commodities dictate the price of aquafeeds: fishmeal and soymeal (Rust et al., 2011; Smith, 2016). In the case of fishmeal, availability is in decline due to ocean overfishing and climate change (Tacon and Metian, 2008; Costello et al., 2012; Cheung et al., 2013). Producing soy, which is also used directly for human consumption, requires large amounts of water (about 2.3 metric tons of water per kg soy beans) (Rust et al., 2011; Mekonnen and Hoekstra, 2011; 2012). These factors along with rising world population lead to a pressing need to find sustainable aquafeed ingredient substitutes. Taking into account alternative natural resources, it has been forecasted that if new ingredient substitutions enter the feed industry, it should be possible to reestablish balance between the production and demand of aquafeeds in the next 25 years (Rust et al., 2011).

[0005] Algae biomass provides an alternative protein source for feeds because algae can partially replace fishmeal and soymeal in animal diets (Toyomizu et al., 2001; Moraine et al., 2004; Kiron et al., 2012). Algae biomass is also rich in other nutrients, vitamins and minerals, making it a promising high-quality aquafeed amendment (Becker, 2004; 2007; Gouveia et al., 2008; Benemann, 2013). However, while recirculated water systems can produce high algae biomass yields with good water efficiency, the cost of producing algae biomass by current technologies remains too high for widespread feed applications (Coutteau and Sorgeloos, 1992). For example, microalgae consortia sells at ~ $3-32 kg ^"1 of dry weight (DW) and Spirulina is ~ $6-7 kg ^"1 DW, while fishmeal is « $1.6-2 kg ^"1 , soy meal « $0.3-0.5 kg ^"1 and fish feed is « $1-2 kg ^"1 . Raceway, open pond systems appear suitable for the production of low-cost algae commodities, but product quality is difficult to maintain (Benemann, 2013). Clearly, a different approach is needed if algae biomass is to become a sustainable aquafeed component.

[0006] Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.

3. SUMMARY

[0007] Apparatuses, systems and methods for growing algae biomass are provided.

[0008] An apparatus for growing algae biomass (also referred to herein as a Sustainable Algae Floe with Recirculation (SAFR) apparatus") is provided, the apparatus comprising:

at least one Algae Growth Raceway (AGR);

an Algae Growth Medium (AGM) reservoir functionally connected to the AGR, at least one AGM flow disrupter positioned in the AGR; and

an AGM circulation system (e.g., pump) for circulating AGM through the at least one

AGR.

[0009] In an embodiment, the apparatus further comprises at least one filter or at least one filtration system disposed in a fluid flow path of the circulating AGM. In an embodiment, the apparatus further comprises an aeration system.

[0010] In an embodiment of the apparatus, the aeration system is positioned in the AGM reservoir.

[0011] In an embodiment of the apparatus, the AGM circulation system collects and recirculates AGM.

[0012] In an embodiment, the apparatus comprises a plurality of algae growth raceways (AGRs), wherein the AGRs are connected in sequence or in parallel.

[0013] In an embodiment of the apparatus, the Algae Growth Raceway (AGR) comprises: an algae trough, wherein at least one algae growth medium (AGM) flow disrupter is disposed in the algae trough;

an inflow system (e.g., a channel, tube, valve, hole, or port);

an outflow (discharge) system (e.g., a channel, tube, valve, hole, or port); and a lid (e.g., a top or sealing portion) for sealing the AGR.

[0014] In an embodiment, the AGR further comprises an algae collecting portion.

[0015] In an embodiment of the apparatus, the algae collecting portion of the AGR is disposed on the surface of the algae trough to keep algae in place for growing.

[0016] In various embodiments, the algae trough can comprise grooved portions, indentations or depressions in which the algae are retained and grow.

[0017] In an embodiment, the apparatus further comprises algae growth medium (AGM), wherein the AGM is stored in the AGM reservoir.

[0018] In an embodiment, the apparatus further comprises a water management system, wherein the water management system:

monitors AGM flow,

monitors a parameter, wherein the parameter is an AGM chemistry parameter or an environmental parameter, or

adjusts the parameter to a desired value.

[0019] In an embodiment of the apparatus, the AGM chemistry parameter or the environmental parameter is pH, ammonium content, nitrate content, nitrite content, phosphate content, oxygen content, redox potential, temperature, light intensity, light periodicity, salinity or AGM flow rate.

[0020] In an embodiment, the apparatus further comprises a sterilization system.

[0021] In an embodiment, the apparatus further comprises a mineralization system or mineralization tank.

[0022] An Algae Growth Raceway (AGR) is provided comprising:

an algae trough, wherein at least one algae growth medium (AGM) flow disrupter is disposed in the algae trough;

an inflow system (e.g., a channel, tube, valve, hole, or port);

an outflow (discharge) system (e.g., a channel, tube, valve, hole, or port); and a lid (e.g., a top or sealing portion) for sealing the AGR. [0023] In an embodiment, the algae growth raceway (AGR) has a regulated flow-rate of AGM.

[0024] In an embodiment, the AGR further comprises an algae collecting portion.

[0025] In an embodiment, the algae collecting portion is disposed on the surface of the algae trough of the AGR to keep algae in place for growing.

[0026] In various embodiments, the algae collecting portion can comprise grooved portions, indentations or depressions in which the algae are retained and grow.

[0027] A system for growing algae biomass (also referred to herein as a "SAFR system") is provided comprising:

a SAFR apparatus; and

algae growth medium (AGM).

[0028] In an embodiment, the system further comprises algae.

[0029] In an embodiment of the system, the algae comprise Scenedesmus spp. (e.g., Scenedesmus quadricauda; S. acuminatus; S. spinosus), Pediastrum spp. (e.g., Pediastrum duplex; P. tetra), Planctonema spp., Monoraphidium spp. or Ankistrodesmus spp.

[0030] In an embodiment of the system, AGM is stored in the AGM reservoir and circulated in the apparatus by the AGM circulation system.

[0031] In an embodiment of the system, AGM comprises (or consists of) fresh water, salt water, seawater, waste water, gray water, recycled water, or eutrophic water.

[0032] In an embodiment of the system, the AGM is supplemented with an AGM

supplement.

[0033] In an embodiment of the system, the AGM supplement is compost tea, fermentation leachate, silage leachate, leachate from anaerobic digestion, leachate from decaying algae biomass, liquid distillation residue, beer-making liquid residue, food waste liquid residue, liquid ejecta of animal farming, microbial sludge (e.g., from a water treatment facility), liquid byproduct of an aquaculture facility, a mineral that supports or augments algae growth, a mineral blend that supports or augments algae growth, or a mineral solution that supports or augments algae growth, or a combination thereof.

[0034] A method for growing algae biomass is provided comprising:

providing a SAFR system, i.e., a system comprising a SAFR apparatus and algae growth medium (AGM), wherein the AGM is contained in or collected in the AGM reservoir of the apparatus; providing an algae culture;

flowing AGM from the AGM reservoir to the AGR of the apparatus;

introducing the algae culture into the AGM;

illuminating the system with natural or artificial light; and

growing algae from the algae culture to produce algae biomass.

[0035] In an embodiment of the method, AGM is recirculated back to the AGM reservoir from the AGR.

[0036] In an embodiment, the method comprises harvesting (or recovering) algae biomass from the system.

[0037] In an embodiment, the harvesting comprises:

draining (or removing) the AGM from the AGR in the system; and

washing the algae biomass from the system (e.g., by increasing velocity of AGM flow, or by using sprinkler systems or high pressure washing systems).

[0038] In an embodiment, the method further comprises collecting or removing AGM from the apparatus.

[0039] In an embodiment, the collecting comprises pumping the AGM from the apparatus.

[0040] A method for remediating an aqueous solution is provided comprising:

providing a SAFR apparatus;

providing an aqueous solution to be remediated, wherein the aqueous solution is an algae growth medium (AGM);

growing algae in the apparatus, and

separating the aqueous solution from algae in the apparatus.

[0041] In an embodiment, the method for remediating further comprises collecting or removing the aqueous solution from the apparatus.

[0042] In an embodiment of the method for remediating, the collecting comprises pumping the aqueous solution from the apparatus.

[0043] In an embodiment of the method for remediating, algae are already present or growing in the aqueous solution to be remediated (i.e., no algae culture is introduced deliberately).

[0044] In an embodiment of the method for remediating, the method further comprises recovering algae biomass from the apparatus. [0045] In an embodiment of the method for remediating, the method further comprises growing a desired species of algae in the aqueous solution in the SAFR apparatus.

[0046] The method can be used to produce an accumulation of algae biomass in a recirculated water system.

[0047] The method can be also used for cleanup of wastewater and the removal of nutrients (e.g., nitrogen and phosphorus) from eutrophicated aquifers and aquaculture systems.

[0048] Algae biomass grown in a SAFR system can be harvested and converted into single- cell protein. Both the algae biomass and the single-cell protein may be used in animal feeds.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Embodiments are described herein with reference to the accompanying drawings, in which similar reference characters denote similar elements throughout the several views. It is to be understood that in some instances, various aspects of the embodiments may be shown exaggerated, enlarged, exploded, or incomplete to facilitate an understanding of the invention.

[0050] FIG. 1. Diagram of an embodiment of a Sustainable Algae Floe with Recirculation (SAFR) apparatus used in an embodiment of a SAFR method to produce algae biomass. When algae growth medium (AGM; in this diagram indicated as gray shading in the AGM reservoir) is added to the SAFR apparatus, this is referred to herein as a "SAFR system." In this embodiment of the SAFR apparatus, the SAFR apparatus comprises at least one Algae Growth Raceway (AGR, also referred to herein as an "algae growth bed"); an Algae Growth Medium (AGM) reservoir functionally connected to the AGR (e.g., fluidically connected, connected directly or indirectly by tubing, conduits or channel(s)) to the AGR), at least one AGM flow disrupter positioned in the AGR; and an AGM circulation system (e.g., pump) for circulating AGM through the at least one AGR. In this embodiment, the AGM reservoir comprises an Aeration System (indicated in the AGM reservoir as a tube producing bubbles). This embodiment of the SAFR apparatus also at least one filter or at least one filtration system in the AGM circulation path ("Filter #1 and Filter #2"). In this embodiment of the SAFR method, algae floe biomass is harvested (in this diagram, indicated as "algae slurry"). The algae slurry is recovered or concentrated (e.g., filtration, sieving, centrifugation, chemical or electrical precipitation) to produce an algae cake. The concentrated algae ("algae cake") is dried ("dry algae biomass") and converted into single-cell protein (a product enriched in protein and with increased digestibility). Both algae biomass and single-cell protein may be used in animal feeds. [0051] FIG. 2. Diagram of a lab-scale SAFR apparatus and SAFR system (SAFR apparatus plus AGM) to grow microalgae floe. In this embodiment of the SAFR apparatus, the AGM reservoir containing AGM (in this diagram indicated as gray shading in the AGM reservoir) is a bottle comprising an aeration system (indicated by bubbles, system not shown). The direction of AGM flow in tubing into and out of the AGM reservoir and AGR is indicated by solid arrows. In this embodiment, the AGR is also a bottle, in which a plurality of glass beads serve as AGM flow disrupters. AGM flow direction in the AGR is indicated by the dashed arrow. In this embodiment, the AGM circulation system or AGM collection and recirculation apparatus is a peristaltic pump. In this embodiment, a filter is interposed between the peristaltic pump and a UV-sterilization system. This embodiment of a lab-scale SAFR system is illuminated by artificial light above and below the AGR.

[0052] FIG. 3. Photograph of a medium-scale embodiment of a SAFR apparatus and SAFR system (SAFR apparatus and AGM) comprising one 1.6 m ² AGR, one 1 m ³ AGM reservoir and one 200 L AGM collection tank that also serves as an AGM recirculation tank ("AGM collection and recirculation"). In another embodiment, the SAFR apparatus shown in this figure can comprise up to six 2.8 m ² AGRs connected serially.

[0053] FIG. 4. Detailed diagram (longitudinal side image) of an embodiment of an Algae Growth Raceway (AGR), a component of a SAFR apparatus.

(1) Water discharge valve #1 (2-3 L/min)

(2) Slurry release valve #1 (20-30 L/min)

(3) Discharge pipe (minimum 1-inch diameter)

(4) Split pipe

(5) Water discharge valve #2 (20-100 ml/min)

(6) Slurry release valve #2 (10-20 L/min)

(7) Algae trough outer layer (Fiberglass, acrylonitrile butadiene styrene, TEFLON®

(polytetrafluoroethylene (PTFE), stainless steel, ethylene propylene diene monomer (EPDM) or polyethylene; 2.4 m L; 1.2 m W; 15 cm H)

(8) Large discharge channel (6-7 cm wide; 3-4 cm deep)

(9) Water (AGM) flow disrupters (for algae floe gravity traps) (10 cm high; 1 cm thick)

(10) Inter- disrupter connectors (for algae floe gravity traps) (1 cm distant from bottom; 8 cm high) (11) Internal liner (stainless steel, or fiberglass gel, or anti-biofilm paint; or non-stick food grade surface such as Fiberglass, acrylonitrile butadiene styrene, TEFLON® (polytetrafluoroethylene (PTFE)), stainless steel, ethylene propylene diene monomer (EPDM) or polyethylene

(12) Connector to uphill algae trough (rubber ring with screw collars)

(13) Lid (can be same material and thickness as (7)); (2.4 m L; 1.2 m W; 30 cm H)

(14) Water jet guidance screw (2.4 m long; 1-2 cm diameter)

(15) Guidance screw holder

(16) Water jet shower head (high pressure shower nozzle)

(17) Water jet mounting piece

(18) High pressure water hose supplying water from a water source (e.g., the AGM reservoir, recycled water or rain water stored in an additional reservoir (distinct from the AGM reservoir), tap water, or any other source of water).

(19) Water inlet coupling valve

(20) Transparent lid (polycarbonate)

[0054] FIGS. 5A-5E. 5A. Top view of another embodiment of an algae growth raceway (AGR). 5B. Side view along the length of this embodiment of an AGR. 5C. A perspective view of this embodiment of an AGR. 5D. Another perspective view of this embodiment of an AGR. 5E. Yet another perspective view of this embodiment of an AGR. The labelling of FIGS. 5A-5E shows the correspondence between the elements of the embodiment of the AGR in FIG. 4 and the elements in the embodiment shown in FIGS. 5A-5E.

5. DETAILED DESCRIPTION

[0055] Current technologies for growing algae focus on providing algae biomass for use in high-value end products (i.e., pharmaceuticals, cosmetics, biofuels, antioxidants, pigments, enzymes, etc.). Otherwise, these current technologies would not be economically viable. Low- cost apparatuses, systems and methods for growing algae to produce biomass are provided herein that are economically viable for use in low- value as well as high- value end products.

[0056] Apparatuses, systems and methods are provided for growing algae biomass are provided. The apparatuses, systems and methods can be employed for algae farming that can be used for producing aquafeeds or for remediation of water sources. These apparatuses, systems and methods produce feed-grade microalgae biomass in a closed raceway at low cost (< $1 kg ^"1 DW). [0057] The method comprises providing a "Sustainable Algae Floe with Recirculation" (SAFR) apparatus and growing aggregated microalgae floe in the SAFR apparatus. In one embodiment, a method is provided for growing tychoplankton (unicellular algae) that live in an aggregate state. By living in an aggregate state, tychoplankton avoid being washed away, but in the aggregate state, they do not stick to surfaces and thus can be easily detached and harvested when desired. In an embodiment of the method, self- aggregated consortia (algae floe) dominated by green microalgae are grown in the SAFR apparatus with 100% recirculated water.

[0058] The methods for growing algae provided herein are referred to herein as Sustainable Algae Floe with Recirculation (SAFR) methods. One embodiment for carrying out the SAFR method is shown in FIG. 1. A SAFR apparatus is provided that comprises at least one Algae Growth Raceway (AGR); an Algae Growth Medium (AGM) reservoir functionally connected to the AGR, at least one AGM flow disrupter positioned in the AGR; and an AGM circulation system (e.g., pump) for circulating AGM through the at least one AGR. The SAFR system for growing algae biomass comprises the SAFR apparatus and algae growth medium (AGM). Algae floe biomass is harvested and converted into single-cell protein (a product enriched in protein and with increased digestibility). Both the algae biomass and single-cell protein may be used in animal feeds. Other potential applications for the SAFR method are cleanup of wastewater and the removal of nutrients (nitrogen and phosphorus) from eutrophicated aquifers and aquaculture systems.

[0059] The SAFR apparatus, system and methods disclosed herein allow for algae biomass to be harvested in an economically viable system to access low- value commodity markets such as animal feed amendments. To accomplish this, the SAFR apparatuses, systems and methods provide more efficient and economical algae cultivation, for example in biomass accumulation and harvesting.

[0060] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections set forth below.

[0061] SAFR Apparatus

[0062] An apparatus for growing algae biomass, referred to herein as a "Sustainable Algae Floe with Recirculation" (SAFR) apparatus is provided. In one embodiment, the SAFR apparatus comprises:

at least one Algae Growth Raceway (AGR); an Algae Growth Medium (AGM) reservoir functionally connected to the AGR, at least one AGM flow disrupter positioned in the AGR; and

an AGM circulation system (e.g., pump) for circulating AGM through the at least one

AGR.

[0063] When algae growth medium (AGM, discussed below) is added to, or included with, the SAFR apparatus, this system is referred to herein as a SAFR system.

[0064] Although many of the embodiments of the SAFR apparatuses, SAFR systems, and SAFR methods disclosed herein involve recirculation of algae growth medium (AGM), as designated by the "R" in the acronym "SAFR," in some embodiments, e.g., in embodiments in which algae growth raceways (AGRs) are connected in sequence, no recirculation takes place. For example, certain AGMs, such as pond water or other water desired to be cleaned or remediated, can be cleaned or remediated by SAFR apparatuses, SAFR systems and SAFR methods that involve several AGRs connected in sequence with no recirculation of AGM taking place. Clean or remediated AGM is produced downstream of the AGRs connected in sequence, without requiring recirculation of the AGM through the AGRs again.

[0065] In another embodiment, clean or remediated AGM is produced downstream of a single AGR or a plurality of AGRs connected in parallel, and in certain embodiments, without requiring recirculation of the AGM through the AGR(s).

[0066] Different scales of SAFR apparatuses can be built. In one embodiment, a small-scale (or lab-scale) SAFR apparatus has the dimensions of about 1 m x 1 m x 0.18 m for the AGR and l m x l m x l m for the AGM reservoir. FIG. 2 shows an embodiment of a small-scale SAFR apparatus and SAFR system.

[0067] In another embodiment, a medium-scale SAFR apparatus has the dimensions of 1 m x 2 m x 0.18 m for the AGR and about 1 m x 1 m x 1 m for the AGM reservoir. FIG. 3 shows an embodiment of a medium-scale SAFR apparatus and SAFR system.

[0068] In another embodiment, a large-scale SAFR apparatus has the dimensions of 1.2 m x 2.4 m x 0.18 m for the AGR and about 1 m x 1 m x 1 m for the AGM reservoir.

[0069] Larger storage volumes can be used for AGM reservoirs and such volumes can be calculated by the ordinarily skilled artisan. The volume of an AGM reservoir is generally dictated by how rapidly AGM flows through the SAFR apparatus. [0070] In another embodiment, several small-, medium- or large-scale system can be combined or many AGRs can be connected in series or in parallel fed from a larger AGM reservoir or from a plurality of AGM reservoirs.

[0071] Algae Growth Raceway (AGR)

[0072] The SAFR apparatus comprises at least one algae growth raceway (AGR), also referred to herein as an "algae growth bed," which is functionally connected to an AGM reservoir. The functional connection from the AGM reservoir to the AGR can be a fluidic connection, e.g., via at least one tube, pipe, conduit, or channel (such as an enclosed channels).

[0073] An AGR can be made of any suitable material known in the art, such as concrete, wood, metal, plastic, or fiberglass. An AGR can be treated or lined on its inside or inner surface with non-stick food grade materials such as fiberglass, acrylonitrile butadiene styrene,

TEFLON® (polytetrafluoroethylene (PTFE)), stainless steel, ethylene propylene diene monomer (EPDM), or polyethylene. In various embodiment, the AGR is substantially or entirely transparent or translucent, or the AGR comprises transparent or translucent portions (e.g., windows), that allow for light penetration (natural or artificial) of the AGR from top and/or bottom and/or sides.

[0074] In an embodiment, the algae growth raceway (AGR) has a regulated flow-rate of AGM. In another embodiment, the SAFR apparatus comprises a regulator to regulate the flow- rate of AGM. Regulators to regulate the flow of fluid in or through an aqueous culture are well known in the art. The flow-rate selects for algal communities that self-aggregate and sink and can be brought in suspension by turbulence (i.e., tychoplankton).

[0075] In an embodiment of the SAFR apparatus, a plurality of AGRs are connected in serial or in parallel to the AGM reservoir.

[0076] In an embodiment, the AGR allows for easy harvesting of algal biomass by comprising at least one algae collection portion (also referred to herein as a "biomass trapping mechanism"). An algae collection portion can be, for example, a groove, channel, indentation, sieve or other collecting portion known in the art. In an embodiment, it disposed on the surface of the AGR.

[0077] The SAFR apparatus can further comprise an algae collecting system (for example, hoses or a high-pressure washer). The algae collecting system periodically disrupts AGM flow and disrupts the attachment of the algae. Algae are suspended and float into an outflow

(discharge) system in the AGR to be collected downstream.

[0078] In an embodiment, the Algae Growth Raceway (AGR) comprises:

an algae trough, wherein at least one algae growth medium (AGM) flow disrupter is disposed in the algae trough;

an inflow system (e.g., a channel, tube, valve, hole, or port);

an outflow (discharge) system (e.g., a channel, tube, valve, hole, or port); and a lid (e.g., a top or sealing portion) for sealing the AGR.

In various embodiments, the algae trough can comprise grooved portions, indentations or depressions in which the algae are retained and grow. The lid can be used to maintain a consistent culture of algae and can help prevent and decrease contamination with outside material in the environment such as leaves and pollen.

[0079] AGM height in the AGR can be controlled and optimized for the growth of tychoplankton. Devices for controlling fluid height in channels or raceways are known in the art.

[0080] FIG. 4 shows a detailed diagram (longitudinal side image) of an Algae Growth Raceway (AGR) from a SAFR apparatus. Shown in FIG. 4 are:

(1) Water discharge valve #1 (2-3 L/min)

(2) Slurry release valve #1 (20-30 L/min)

(3) Discharge pipe (min 2 inches diameter)

(4) Split pipe

(5) Water discharge valve #2 (20-100 ml/min)

(6) Slurry release valve #2 (10-20 L/min)

(7) Algae trough outer layer (Fiberglass or acrylonitrile butadiene styrene or

polyethylene; 2.4 m L; 1.2 m W; 15 cm H)

(8) Large discharge channel (6-7 cm wide; 3-4 cm deep)

(9) Water (AGM) flow disrupters (for algae floe gravity traps) (10 cm high; 1 cm thick)

(10) Inter- disrupter connectors (for algae floe gravity traps) (1 cm distant from bottom; 8 cm high)

(11) Internal liner (stainless steel, or fiberglass gel, or anti-biofilm paint; or TEFLON® (polytetrafluoroethylene (PTFE))

(12) Connector to uphill algae trough (rubber ring with screw collars)

(13) Lid (same material and thickness as (7)); (2.4 m L; 1.2 m W; 30 cm H) (14) Water jet guidance screw (2.4 m long; 1-2 cm diameter)

(15) Guidance screw holder

(16) Water jet shower head (high pressure shower nozzle)

(17) Water jet mounting piece

(18) High pressure water hose

(19) Water inlet coupling valve

(20) Transparent lid (polycarbonate)

[0081] FIGS. 5A-5E show diagrams of another embodiment of an AGR. FIG. 5A is a top view. FIG. 5B is a side view along the length of this embodiment of an AGR. FIG. 5C is a perspective view of this embodiment of an AGR. FIG. 5D is another perspective view of this embodiment of an AGR. FIG. 5E is yet another perspective view of this embodiment of an AGR. The labelling of FIGS. 5A-5E shows the correspondence between the elements of the embodiment of the AGR in FIG. 4 and the elements in the embodiment of the AGR shown in FIGS. 5A-5E.

[0082] AGM reservoir

[0083] The SAFR apparatus further comprises an AGM reservoir that contains or stores the AGM and is in fluidic communication or is fluidically connected to the AGR to supply AGM to the growing algae. The AGM reservoir can be connected to, or feed into, the AGR, by any means known in the aquaculture art, for example, fluidically (e.g., via an enclosed channel, tube, pipe, conduit, or hose).

[0084] In an embodiment, the AGM reservoir is sealed to prevent environmental contamination, with no light penetration or air penetration. The SAFR apparatus can also comprise a lid that seals the AGM reservoir. For example, the lid can comprise a gasket(s) (e.g., rubber gasket) for sealing the AGM reservoir. In an embodiment, the AGM reservoir is opaque and dark in color, to discourage or avoid algae growth in the AGM reservoir or in other parts of the SAFR apparatus.

[0085] The AGM reservoir can range in volume depending on the size of the SAFR apparatus contemplated, and a suitable size for the AGM reservoir in the SAFR apparatus can be calculated using methods known in the art. As mentioned above, an embodiment of a small- scale (lab-scale) or medium-scale SAFR apparatus can have an AGM reservoir of about 1 m x 1 m x 1 m. FIG. 2 shows an embodiment of a small-scale SAFR system and FIG. 3 shows an embodiment of a medium-scale SAFR system with AGM reservoirs that can have these dimensions.

[0086] In another embodiment, a large-scale SAFR apparatus can have an AGM reservoir of about l m x l m x l m.

[0087] AGM flow disrupter

[0088] The SAFR apparatus comprises at least one AGM flow disrupter (or AGM flow barrier) positioned in the AGR. In embodiments, the AGM flow disrupter(s) may consist of glass beads, pebbles, rods, depressions, unevenness of the AGR acting as algae traps, or other material disrupters or barriers added to the AGR or built within the AGR. The AGM flow disrupter is used to disrupt laminar flow of the AGM and allow algae floe to settle.

[0089] AGM circulation system

[0090] The SAFR apparatus comprises an AGM circulation system or AGM collection and recirculation apparatus, e.g., a water pump. Circulation systems for circulating water or other fluids, fluid or water collection and recirculation apparatuses and/or fluid or water pumps can be used and are well known in the art. In one embodiment of the SAFR apparatus, the AGM circulation system collects and recirculates AGM. In another embodiment of the SAFR apparatus, the AGM circulation system does not collect and/or recirculate AGM.

[0091] In embodiments, the AGM circulation system SAFR apparatus can recirculate AGM to conserve water or to recycle nutrients to reduce costs.

[0092] Additional elements that can be used in SAFR apparatuses, SAFR systems and SAFR methods

[0093] In an embodiment, the SAFR apparatus comprises at least one filter or at least one filtration system at least one filter or at least one filtration system disposed in a fluid flow path of the circulating AGM.

[0094] In an embodiment, the filtration system filters out particulate matter (e.g., particles at least 10 microns in diameter). The filtration system can, in certain embodiments, host microbes to mineralize organic matter.

[0095] In an embodiment, the SAFR apparatus comprises an aeration system. Suitable aeration systems are well known in the art. In an embodiment, the aeration system is positioned in or comprised in the AGM reservoir. In another embodiment, the aeration system aerates the AGM in the AGM reservoir. In another embodiment, aeration of the AGM by the aeration system occurs outside of the AGM reservoir

[0096] In an embodiment, the SAFR apparatus further comprises a water management system. In embodiments, the water management system monitors physical fluid flow of the AGM, monitors AGM chemistry parameters or environmental parameters, and / or adjusts an AGM chemistry parameter or an environmental parameter of the SAFR apparatus to desired values. Such AGM chemistry parameters or environmental parameters can include, but are not limited to, pH, ammonium content, nitrate content, nitrite content, phosphate content, oxygen content, redox potential, temperature, light intensity, light periodicity, salinity and AGM flow rate. Water management systems for physical and chemical monitoring of water and adjustment of water chemistry and environmental parameters are well known in the aquaculture art.

[0097] In an embodiment, the SAFR apparatus further comprises a sterilization system. The sterilization system (for example, based on UV, ozone, steam, microwave, pasteurization or other means of sterilization) is used to clean the SAFR apparatus before inoculation with desired algae, to sterilize the water in the system, or to select against planktonic algae.

[0098] In an embodiment, the SAFR apparatus comprises a mineralization system or a mineralization tank. Mineralization tanks and systems are known in the art. In an embodiment, the AGM reservoir also serves as a mineralization tank.

[0099] In an embodiment, the SAFR apparatus further comprises an AGM collection tank and/or an AGM recirculation tank. In an embodiment, the AGM collection tank also serves as an AGM recirculation tank. In another embodiment, the AGM reservoir also serves as an AGM collection tank and/or an AGM recirculation tank.

[00100] In various embodiments, the SAFR apparatus can comprise any of the following elements known in the aquaculture art to increase the efficiency of using the SAFR apparatus for producing algae biomass: a variable water flow regulator for regulating the flow of AGM, a controller of the aerator, or a switch system between day and night for using solar energy with increased efficiency.

[00101] In another embodiment, the SAFR apparatus comprises microbial fuel cell(s), systems or devices for ozonation, systems or devices for anaerobicity, and/or biofilms. Such systems and devices are well known in the art. [00102] Algae Growth Medium (AGM) and AGM supplements for use in SAFR apparatuses, SAFR systems and SAFR methods

[00103] In an embodiment, the SAFR apparatus further comprises algae growth medium (AGM), which is any medium in which algae will grow. This embodiment is also referred to herein as a "SAFR system," i.e., a system comprising a SAFR apparatus and algae growth medium (AGM).

[00104] AGM can be a natural solution or a synthetic solution. In certain embodiments, the AGM is a natural solution. Any natural solution known in the art to support the growth of desired algae species may be used, e.g., fresh water, salt water, seawater, waste water, gray water, tap water, recycled water, eutrophic water from natural systems.

[00105] In other embodiments AGM is a synthetic aqueous solution comprising minerals. Any synthetic aqueous mineral solution known in the art to support the growth of desired algae species may be used, e.g., standard mineral solutions known in the art for growing algae. Such mineral solutions can comprise ions and macro nutrients such as ammonium, nitrate, phosphate, carbonate, potassium, sodium and essential micronutrients; the identities and concentration of nutrients are variable depending on the various species grown. Such nutrients and the concentrations needed to support algae growth are known in the art. AGM supplements can also comprise minerals; ions or macro nutrients such as ammonium, nitrate, phosphate, carbonate, potassium, or sodium; or essential micronutrients that support the growth of algae.

[00106] In certain embodiments, AGM is supplemented or amended with an AGM supplement. AGM supplements or amendments can include, but are not limited to, compost tea, fermentation leachate, silage leachate, leachate from anaerobic digestion, leachate from decaying algae biomass, liquid distillation residue, beer-making liquid residue, food waste liquid residue, liquid ejecta of animal farming, microbial sludge (e.g., from a water treatment facility), liquid byproduct of an aquaculture facility, a mineral that supports or augments algae growth, a mineral blend that supports or augments algae growth, and a mineral solution that supports or augments algae growth, or a combination thereof.

[00107] AGM can be supplemented or modified by adding at least one of the above-listed AGM supplements or amendments. For example, AGM (e.g., recycled water) can be supplemented with a fermentation leachate, e.g., at 1 part fermentation leachate per 100 parts AGM. Algal growth supplements or amendments can be used to trigger algae biomass growth or to limit the growth of undesirable algae. [00108] In one embodiment, AGM supplement can be supplied by direct addition (e.g., pouring in) to the SAFR system or by feeding in the AGM supplement into the SAFR system from an additional reservoir for AGM supplement in the SAFR apparatus. In one embodiment, the additional reservoir for AGM supplement is fluidically connected to the flow path of AGM in the SAFR apparatus.

[00109] Algae cultures for use in SAFR apparatuses, SAFR systems and SAFR methods

[00110] Algae suitable for producing biomass may be cultured (or grown) in the SAFR apparatus according to the methods disclosed herein. Algae communities or cultures that can grow in the SAFR apparatus include, but are not limited to loose microalgae floe (also known as tychoplankton), algae biofilm on substrates, floating algae mats, filamentous algae and phytoplankton. In one embodiment, loose microalgae floe (also known as tychoplankton) is grown in the SAFR apparatus.

[00111] In one embodiment, an algae culture is provided for growing in the SAFR apparatus in which the predominant species is Scenedesmus spp. (e.g., Scenedesmus quadricauda; S. acuminatus; S. spinosus), Pediastrum spp. (e.g., Pediastrum duplex; P. tetra), Planctonema spp., Monoraphidium spp. or Ankistrodesmus spp.

[00112] The SAFR apparatus may be used to grow algae aggregates that are loose, sinking, or not attached to surfaces. At least five types of algae communities can be grown in SAFR apparatuses, including but not limited to: loose microalgae floe (sinking aggregates); biofilm (algae-rich layers attached to abiotic substrates); floating mats (algae masses at the water's surface); filamentous algae with "cotton candy" appearance (often attached to substrates); and phytoplankton (single cells and small aggregates in suspension).

[00113] Algae cultures for use in SAFR apparatuses, SAFR systems and SAFR methods are further discussed below in "Methods for Growing Algae Biomass."

[00114] Method for Growing Algae Biomass

[00115] A method for growing algae biomass is provided. The method for growing algae biomass can be used to produce an accumulation of algae biomass in a recirculated or a non- recirculated water system. In an embodiment of the method, algae biomass is grown in the SAFR apparatus with 100% recirculated water. [00116] In an embodiment, the method for growing algae biomass comprises providing a SAFR apparatus and growing algae in the SAFR apparatus. In an embodiment, the algae are aggregated microalgae floe or self- aggregated consortia ("algae floe") dominated by green microalgae.

[00117] The method for growing algae biomass can be also used for cleanup of wastewater and the removal of nutrients (e.g., nitrogen and phosphorus) from eutrophicated aquifers and aquaculture systems.

[00118] In an embodiment, the method for growing algae biomass comprises providing an SAFR apparatus and growing aggregated microalgae floe dominated by the green algae Scenedesmus quadricauda in the SAFR apparatus as disclosed herein. Growing the aggregated microalgae floe in a SAFR apparatus with a recirculated algae growth medium (AGR) system allows accumulation of the algae biomass (see embodiment of SAFR apparatus shown in FIG. 1). The algae biomass grows from AGM nutrients and energy received from light illumination.

[00119] In an embodiment, the method for growing algae biomass comprises:

providing a SAFR system as disclosed herein, comprising a SAFR apparatus and algae growth medium (AGM), wherein the AGM is contained in or collected in the AGM reservoir of the SAFR apparatus;

providing an algae culture;

flowing AGM from the AGM reservoir to the AGR of the SAFR apparatus; introducing the algae culture into the AGM;

illuminating the system with natural or artificial light; and

growing algae from the algae culture to produce algae biomass.

[00120] In an embodiment of the method, AGM is recirculated back to the AGM reservoir from the AGR.

[00121] In certain embodiments, AGM can be tested and / or amended in the SAFR apparatus.

[00122] In certain embodiments, AGM can be mineralized in the SAFR apparatus, e.g., in the AGM reservoir.

[00123] In certain embodiments, AGM can be filtered in the flow path of the AGM in the SAFR apparatus, e.g., in the flow path from the AGM reservoir to the inflow of the AGR, or in the flow path from the outflow of the AGR and the AGM reservoir. [00124] Methods for harvesting algae from the SAFR apparatus are also provided. In one embodiment, the method comprises:

providing a SAFR system and algae biomass grown in the SAFR system to be harvested, draining (or removing) the AGM from the AGR in the SAFR system; and

washing the algae biomass from the SAFR system, for example, by increasing velocity of AGM flow, or by using sprinkler systems or high pressure washing systems.

[00125] In certain embodiments, algae biomass may or may not be allowed to settle before it is decanted or removed from AGM and from the SAFR system. In other embodiments, algae biomass may be removed from AGM by methods well known in the art, such as centrifugation filtration, or chemical or electrical precipitation.

[00126] In one embodiment of the method, AGM is recirculated, nutrients are mineralized and recycled, and algae are cultured and harvested, wherein the algae have a natural tendency to aggregate. At various time intervals, nutrients are added to compensate for nutrients removed by the growing algae biomass.

[00127] In one embodiment, the SAFR apparatus or SAFR system may be attached to an established aquaculture facility and may be used to capture undigested nutrients from wastewater produced by the aquaculture facility. This allows for reclamation of nutrient input and adds more value to the aquaculture facility.

[00128] In another embodiment, the SAFR apparatus or SAFR system can be employed to extract nutrients from liquid byproducts of food waste. The SAFR apparatus and methods may be used to make food waste facilities becoming more efficient and sustainable.

[00129] In another embodiment, a method for remediating an aqueous solution is provided comprising:

providing a SAFR apparatus;

providing an aqueous solution to be remediated, wherein the aqueous solution is an algae growth medium (AGM);

growing algae in the apparatus, and

separating the aqueous solution from algae in the apparatus.

For example, a SAFR apparatus or SAFR system can be employed to extract nutrients from eutrophication-polluted aquatic systems such as rivers, estuaries, wetlands, lakes, water storage facilities, groundwater aquifers. In one embodiment, excess phosphorus and nitrogen can be extracted or removed. [00130] In an embodiment of the method for remediating, algae are already present or growing in the aqueous solution to be remediated (i.e., no algae culture is introduced deliberately).

[00131] The SAFR method optimizes production targeted at loose microalgae floe that sinks, does not attach to surfaces, and is brought in suspension by turbulence. Such communities are classified in limnology as tychoplankton (Poulickova et al., 2008). The tychoplankton community offers several advantages for biomass cultivation: 1) it can be designed to contain one or a combination of known algae; 2) cells auto-flocculate, making them economical to harvest; and 3) with recirculation it consumes very little water. Many factors must be optimized to produce maximum and high-quality algal tychoplankton biomass, including: using verified algae strains, nutrients and nutrient recycling by mineralization, raceway design, fluid flow of AGM, light quality and regime, temperature, aeration and harvesting protocol. The inventors' studies of medium-scale SAFR apparatuses (with ~ 2 m ² AGR, FIG. 3) have produced consistently high yields of algal biomass dominated by Chlorophyceae (~ 90 % in number of cells and ~ 55 % in Vol/Vol) and diatoms (~ 9 % in number of cells and ~ 45% in Vol/Vol). Contaminating Euglenophyceae and Cyanobacteria combined did not amount to more than 0.1 % Vol/Vol (Table 1).

[00132] In one embodiment of the method, microalgae consortia (rather than monocultures) are used to provide robust communities well-suited for producing feed biomass. Consortia make the floe more stable to variation in temperature, light and nutrient availability and better occupy trophic niches (i.e., expected to be more resilient to the growth of algae contaminants). Using a consortium of selected algae also allows multi-season variation of the community structure, yet biomass remains feed-safe. In one embodiment, a loose algae floe consortium comprising nontoxic green algae, such as Scenedesmus, Pediastrum, Planctonema and Monoraphidium, and diatoms are used (see Table 1).

[00133] Scenedesmus quadricauda (a dominant species) is a well-known planktonic alga capable of growing as unicells (Titman and Kilhman, 1976) or as aggregates (Hondzo and Lyn, 1999; Hondzo et al., 1998; Jeon et al., 2013). This alga is nutritious, grows in wide range of nutrient availability and can also grow as a mixotroph, which is important for growth in recycled water systems (Ahlgren and Hyenstrand, 2003; Toyub et al., 2008; Kandimalla et al., 2015). Pediastrum species are coenobial and shown to induce flocculation of other algae as well (Grewe and Markus, 2000; Weckstrom et al, 2010). Planctonema has a filamentous phenotype, expected to help increase the cohesiveness of the floe. Monoraphidium is related to Scenedesmus and participates in aggregates as well (de Queiroz et al., 2012; Jeon et al., 2013).

[00134] Chemical content of cultured algae varies with species composition, media composition, specific treatments and culture age. Single-cell protein preparation from the SAFR algae biomass from Table 1 contains (per DW): ^¾ 32% crude protein; ~ 3% fat; ~ 35% carbohydrates; and ~ 30% ash. The biomass yield is approximately 30 g of DW m ^"2 of AGR day 1 and electrical energy costs are ~ $0.55 kg ^"1 of DW. Approximately 90% of the energy needed may be provided by solar panels, ~ 2% is used for water (AGM) circulation and aeration, ~ 30% for harvesting the algae slurry, ~ 13% for concentrating the slurry by centrifugation, and ~ 56% for drying and post-processing of the biomass into single-cell protein. Water consumption is very low (~ 43 L kg ^"1 algae biomass DW) and production costs < $1 kg ^"1 . Further sizable reduction in costs may be obtained by: selection of algae strains with desirable properties, and by making improvements in system design, growth management and in the post-processing of biomass into feeds.

[00135] Analysis of tychoplankton community grown in medium-scale SAFR

apparatuses. Algae tychoplankton can be analyzed growing in SAFR apparatuses with 1.6-2.8 m ² AGRs (FIG. 1). Routine microscopic evaluation may be used for easy and cost-effective monitoring of the health of the tychoplankton community and overall water quality in the system. For example, approximately ten samples a week may be analyzed for cell density and morphological characterization of the algae cells. This data may be used to identify changes in the community structure and identify algae of interest.

[00136] Isolation of dominant strains of interest and maintain pure cultures. In one embodiment, tychoplankton produced by pure cultures and algae consortia are used. In one embodiment, the genus Scenedesmus is used. Secondary participants are from the genera Pediastrum, Planctonema and Monoraphidium. Isolating strains afford the ability to amend or restock the SAFR AGRs with desirable species or mixtures. Pure cultures also permit the ability to experiment with various proportions of organisms and provide biological contingency in the event of contamination or loss.

[00137] Selection of isolated strains based on optimal traits of chemical composition.

Isolated strains are screened based on chemical features that are important in feeds (crude/true protein, amino acids profile, starch, total fat, fatty acids profile, fiber, ash and toxins. Selection criteria include, but are not limited to: algae strains producing high abundance of true proteins and no ichthyotoxins in tychoplankton state. The second tier of selection criteria is ash content and amino acids and fatty acids profile.

[00138] Characterization of physical properties of cultured tychoplankton. Physical features of the tychoplankton aggregates may be characterized, such as dimension, porosity, relationship between wet biomass and dry weight, sedimentation rate, and attachment to artificial substrates present in AGRs (FIG. 1): borosilicate glass and acrylonitrile butadiene styrene (ABS). These results can give information about aggregation properties of various algae, expected attachment to the SAFR racetrack materials, and help determine how growth conditions influence aggregation and floe buoyancy. This information may be used to optimize the management of SAFR apparatuses and systems.

[00139] Testing strains and consortia for ability to form tychoplankton in lab-scale SAFR systems. Algae strains and algae consortia are tested in lab-scale SAFR apparatuses (FIG. 2) for the ability to grow as tychoplankton (i.e. self-aggregation phenotype), relative to growing as phytoplankton or as surface-attached biofilms. A first-hand screen-test is done on a standard set of specific conditions (with respect to AGM chemistry, illumination regime and flow rate). Then, strains or consortia of interest are tested for forming tychoplankton in response to selected variables.

[00140] Analyses of the effects of physical variables on the evolution of tychoplankton in lab-scale SAFR systems. Conditions are sought that favor the formation of aggregates, and non- attachment to solid substrates (such as glass or ABS). A lab-scale SAFR apparatus may be used to determine these conditions. The independent physical variables are: AGM velocity and depth, light intensity, photoperiod, temperature and density of the AGM solution. The following dependent variables may be monitored: ratio between loose aggregates and cells attached to surfaces or cells in suspension; aggregate size and sedimentation rate and abundance of desirable green algae. Analysis of variance is used to determine most favorable physical conditions for tychoplankton formation in SAFR apparatuses and systems.

[00141] Analyses of the effects of chemical variables on evolution of tychoplankton in lab-scale SAFR systems. A lab-scale SAFR apparatus (FIG. 2) is used to determine optimal chemical parameters for producing tychoplankton. The variables can include nutrient concentrations, N:P ratio, sulphate, pH, hardness, O2 concentration and Biochemical Oxygen Demand (BOD). Ammonium is particularly important because it is correlated with auto- flocculation of the green algae Scenedesmus obliquus and Ankistrodesmus falcatus (Salim et al., 2011). The importance of other factors was discussed above. The dependent variables discussed above, as well as respiration rate, are studied. The analysis of BOD helps to calculate the optimum size and mineralization rate in the reservoir (FIG. 1), and the culture medium turnover time. The measuring of respiration may be used to optimize the relationship between: biomass density, fluid flow rate of AGM at night, oxygen availability and harvesting frequency.

[00142] Monitoring of seasonal evolution of tychoplankton in medium-scale SAFR systems in response to environmental variables. Pre-sterilized medium-scale SAFR apparatuses are inoculated with lab-grown tychoplankton. System variables (flow rate of AGM, temperature, illumination regime, pH, nutrient content, O2 at night and BOD) are monitored. Harvesting tychoplankton at various time intervals determines the optimal harvesting periodicity as a function of other parameters. Changes in O2 concentration between input and output determine risks of night-time anoxia relative to the abundance of tychoplankton and

temperature. Seasonal evolution of biomass yield and community structure is also monitored.

[00143] Production of algae biomass in medium-scale SAFR systems. Outdoor mid-scale SAFR systems (see, e.g., FIG. 3) with 1.6 to 2.8 m ² AGB can then be inoculated with target algae strains and harvested weekly when the tychoplankton abundance reaches about 2.7-3 g L ^"1 . The harvested algae slurry produced (~ 20-30 L m ^~2 AGB) is then allowed to settle in a funnel in the cold room (5°C) for 6-12 hours. Then the top layer is decanted, leading to a thick algae slurry with 2.7-3 % g biomass DW, further concentrated by continuous flow centrifugation into an algae cake with 13-20% biomass DW. The experimental algae biomass yield is then determined.

[00144] Processing algae biomass into various single-cell protein preparations. Harvested algae biomass may be tested in feed trials. Harvested algae biomass can also be also processed into various single-cell protein (SCP) preparations. The SCP preparation methods vary with respect to the chemistry and nutrition objectives. Some methods focus on cell fracturing to increase the digestibility of proteins. Some methods focus on lowering the concentration of toxic metabolites. Some methods focus on partial hydrolysis/denaturing to increase the digestibility of carbohydrates and proteins. Lastly, some methods focus on lowering the concentration of RNA (which in algae is too abundant for feeds).

[00145] Determination of nutrient properties of the algae biomass and single-cell protein. The algae biomass and SCP preparations are analyzed for the following nutrient parameters: dry matter, ash, crude and true protein, crude lipid, RNA, starch, gross energy, minerals, amino acids, fatty acids and crude fiber. Results are compared to the nutritional composition of common feed ingredients such as fish meal, soy bean meal and soy protein concentrate. These measurements may be used to develop feed formulations based on the biomass produced by the SAFR method and system.

[00146] Determination of concentration of specific toxins in algae biomass and single- cell protein. Algae biomass and various SCP preparations are analyzed for the concentration of specific toxic chemicals. Lysinoalanine (a common byproduct of food processing) and cyanotoxins (microcystins, nodularin, cylindrospermopsin, saxitoxins and anatoxin-a) are analyzed. These measurements, in combination with microscopic survey of the community for cyanobacteria and feed trials may be used to verify that the SAFR technology and products are feed-safe and constrain the timing for harvesting, system cleanup, sterilization, re-inoculation with desirable species and chemical treatments.

[00147] Feed trials on insects. Nutritional and other effects of adding microalgae powder, produced by the SAFR method, can be tested in an insect diet, e.g., in the diet of black soldier fly larvae (Hermetia illucens). In experiments, control larvae are fed diets of 100% beer mash and experimental larvae are fed diets in which some of the beer mash is replaced with algae powder. Food conversion ratios are determined using methods known in the art. In actual tests (see Section 6.3), food conversion ratios were obtained ranging between 0.95 and 1.2 with an average of approximately 1.

[00148] In experiments, larvae are fed for 10 days at approximately 25 C and harvested by separation from the growth mixture and washing on the twelfth day. Growth rates (based on triplicates and 1 standard deviation differences) are determined using methods known in the art and compared between control larvae fed control diets and experimental larvae fed diets containing algae powder.

[00149] Feed trials on fish with algae biomass and single-cell protein preparations. A long term feeding trial may be used to evaluate chronic nutritional and other effects of an ingredient or a feed on fish (e.g., tilapia). Two-step feed trials may be conducted to test the potential of the algae biomass and SCP as feed ingredients. First, these two ingredients are tested for the Apparent Digestibility Coefficient (ADC) of major nutrients such as dry matter, protein and gross energy. Based on the results of ADC tests, a series of test diets may be formulated containing the algae biomass or the single-protein at different levels. The algae ingredients are compared to common commercial ingredients (fishmeal and soymeal) regarding their effects on the performances of Mozambique tilapia tested under controlled laboratory conditions. The outcome may be used to estimate the production cost of tilapia using algae ingredient based diets. Because feed represents more than 50% of the production cost of fish, this would have a significant impact on operating expenses for the commercial aquaculture industry.

[00150] Determination of the quality and safety of fish fillet produced using algae-based feed. After the nutritional composition (dry matter, ash, protein and lipid) of fish fillets generated in the fish feeding trials is determined using methods known in the art, this information is used to compare nutrient retentions of different diets and nutritional quality of end products. The fish fillets are also subjected to sensory evaluation to determine whether the ingredients have any adverse effect on market parameters. Specific toxins in the whole fish and different tissues (liver, muscle, and viscera) are also analyzed if any specific algae toxin is identified as described above. These results provide evaluation of the nutritional quality as well as safety of using the algae ingredients for feed production.

[00151] Methodology and Techniques

[00152] Analyzing tychoplankton community from medium-scale SAFR systems. The tychoplankton community is routinely assessed with live specimens using a CKX31 inverted light microscope (Olympus). A subsample from the freshly harvested material is added to a polystyrene dish (Falcon) containing filtered water (AGM) from the same raceway. The reporting scheme is presented as relative abundances of organisms in a given sample: where dominant species represent 50% or more of the sample, abundant (24-49%), common (10-24%), present (1-9%) and rare (< 1%). Another subsample obtained from the same harvested material is preserved with Lugol's iodine and stored in 30 mL borosilicate glass scintillation vials as an archive.

[00153] Isolation of dominant strains of interest and maintain pure cultures.

Dominant photosynthetic members of the tychoplankton are: 1) micropipette isolated and propagated clonally in dilute DY-V freshwater algal medium; 2) added to sterile 24-well tissue culture plates (Costar); and; 3) placed into upright incubators at ambient experimental temperature under medium light illumination (20-300 μΕ m ² sec ^"1 ) under 12h: 12h light:dark cycle. Successful isolates are transferred to tissue culture flasks containing the same medium and established in culture. The culture media for Scenedesmus, Pediastrum, Planctonema,

Monoraphidium and Ankistrodesmus are modifications after Chen et al., (2014). Isolated strains may be identified by 18S rDNA sequencing. This approach has been used previously with many natural samples and has been used to establish an extensive culture collection.

[00154] Selection of isolated strains based on optimal traits with respect to chemical composition. The chemical composition of the algae biomass and the ash content is analyzed using the protocol recommended by Laurens et al., (2012). Protein measurements (crude and true) may be done according to the methods known in the art (e.g., Gonzalez Lopez et al.

(2010)). Cellulose measurements are outsourced and determined by the Acid Detergent Fiber assay. Algal biomass and ash content are determined by calcination (APHA, 2005). Amino acid profiles are determined by High Performance Liquid Chromatography. Fatty acids may be analyzed after derivatization using a gas chromatograph equipped with a flame ionization detector according to the methods of Ju et al. (2012).

[00155] Characterization of physical properties of cultured tychoplankton. Algae aggregate characteristics (length, with, area, perimeter and circularity) are determined after taking images on an inverted microscope with image recording capabilities (Hondzo et al., 1998). The photomicrographs are also used to estimate porosity and biovolume (Wetzel and Likens 1991; Sieracki et al. 1998). Indirect estimation of biovolume is realized by direct quantitation of algal photosynthetic pigments (Hotzel and Croome, 1999). The sinking / sedimentation rate of tychoplankton aggregates is determined by the SETCOL method

(Bienfang, 1981). Adhesion of floe to surfaces is done by gravimetry and chlorophyll content after algae floe and cells are exposed to glass and ABS plastic beads.

[00156] Testing strains and consortia for the ability to form tychoplankton in lab- scale SAFR systems. Algae strains and algae consortia are inoculated in lab-scale SAFR apparatuses. Variables are manipulated (i.e., velocity of AGM flow, light intensity, periodicity) and the relationship measured between tychoplankton relative to algae attached to glass beads, algae attached to ABS plastic beads and algae in suspension. The algae biomass is determined based on gravimetry and chlorophyll content (Hotzel and Croome, 1999). Aggregation is measured by a method derived from Grewe and Markus (2000).

[00157] Analysis of the effect of physical variables on the evolution of tychoplankton in lab-scale SAFR systems. Illumination regime (intensity, photoperiod and photosynthetically active radiation (PAR) in the range 400-700nm) are measured with data logger lux meters (MSR145) and with a Universal Light Meter & Data Logger ULM-500 coupled with Spherical Micro Quantum Sensor US-SQS/L (Riebesell, 1989; Wetzel, 2001; Wetzel & Likens 2001). A video recorder is used to analyze AGM velocity and flow characteristics around glass beads in lab-scale setups with 1% Kalliroscope particles added (Hondzo et al., 1998). AGM density is measured with conventional hydrometers. Tychoplankton sinking and sedimentation rates are measured according to the methodology disclosed herein. Growth rate for the tychoplankton is calculated from sampling SAFR algae cultures or biomass periodically and analyzing dry weight and changes in photosynthetic pigments. Community structure is characterized using a Taxon Diversity Index (based on Shannon and Weaver 1949), Taxon Evenness (based on Pielou, 1969; 1975) and Taxon Richness indices (based on Clifford and Stephenson, 1975; and Whittaker, 1977). For multivariate analysis, the protocol described in Ruma and Choudhury (2014) may be used.

[00158] Analysis of the effect of chemical variables on the evolution of tychoplankton in lab-scale SAFR systems. The effect of chemical variables on the evolution of tychoplankton in lab-scale SAFR systems may be analyzed using methods known in the art. The evolution of the pH is monitored with pH meters with recording capabilities. Temperature and dissolved oxygen may be measured using a YSI Model 57 probe. A separate, YSI Model 33 probe may be used to measure conductivity. Hardness may be measured by titration using a Hach kit (model 5- b). Ammonia may be measured by the Nessler method (modified after Jenkins, 1967). Nitrite and nitrate may be measured by the hydrazine reduction method (modified after Greenberg et al., 1985). Phosphate may be measured by the molybdate-ascorbate method (modified after Chen et al., 1956). Total nitrogen may be measured with a CHN FLASH 2000 CHN instrument. Respiration may be measured based on consumption of O2 in dark BOD bottles.

[00159] Monitoring seasonal evolution of the tychoplankton in medium-scale SAFR systems in response to environmental variables. Mid-scale SAFR systems may be incubated for longer periods (up to 12 months) to determine how the tychoplankton community changes in various seasons without re-inoculating the system. The methods used to measure biomass density, growth rate, environmental and chemical parameters and community structure are disclosed above.

[00160] Production of algae biomass in medium-scale SAFR systems. In tests, medium-scale SAFR systems with 1.6 to 2.8 m ² AGR (FIG. 1) may be used to produce biomass for feed trials. These systems can vary with respect to algae community, nutrient concentration and AGM velocity. For harvesting, the SAFR system is drained of supernatant AGM. The settled tychoplankton is washed off the growth substrate with a high-pressure water hose. This concentrated solution of algae is allowed to separate from the AGM by settling in a cold room at 5°C for 6-12 hours. The resulting algae slurry is concentrated with a continuous flow centrifuge (60 L hr ^"1 ; 6,000 rpm).

[00161] Processing algae biomass into various single-cell protein preparations. In one embodiment, the following method is used to process algae biomass into single-cell protein (SCP).

[00162] - The algae cake is frozen, then thawed and suspended in water to produce a slurry with about 90% water. [00163] - Heat treatment (64 C; 5 min.) to inhibit fungal, algal and bacterial proteases without affecting RNAses.

[00164] - Incubation (30 ^° C; 24 hours) for partial digestion of RNA by cellular RNAses.

[00165] - Heat treatment (~ 98-99 C; 10 min.) to precipitate proteins and increase digestibility.

[00166] - Freeze-thawing the mixture three times to fracture the cells.

[00167] - Concentration by centrifugation, washing and drying at 95 C to constant weight.

[00168] - The hydrophobic fraction (containing fats) from supernatant is saved and used in feed mixtures.

[00169] This treatment is designed to increase the digestibility of proteins and carbohydrates, to decrease biotoxins and to reduce the concentration of RNA, is less likely to form lysinoalanine and saves the algae oil. Using methods known in the art, the method may be improved to obtain better results with respect to energy efficiency, digestibility (Nasseri et al. 2011; Piasecka et al., 2014; Becker, 2004), cell wall hydrolysis with commercial enzymes (Sander and Murthy, 2009; Huo et al 2015) and RNA decrease (Hedenskog and Morgen, 1973).

[00170] Determination of the nutrient properties of algae biomass and single-cell protein (SCP). Proximate composition (dry matter, ash, protein, lipid and fiber) and gross energy of ingredients are analyzed following the method of the AO AC International (2005). See also Section 6.2.

[00171] Minerals are analyzed, for example, using inductively coupled plasma atomic emission spectroscopy. Amino acids are analyzed, for example, by HPLC with fluorescent detection of dabsyl derivatives (Krause, 1995; Castillo and Castells, 2001; Takeuchi, 2004). RNAs are analyzed, for example, by spectrophotometry after extraction and purification (Kim et al., 2012). Fatty acids are analyzed, for example by an external contractor or service that specializes in feed analysis, after derivatization using a gas chromatograph equipped with a flame ionization detector (GC-FID) according to the methods of Ju et al. (2012).

[00172] Determination of concentration of specific toxins in algae biomass and single-cell protein.

[00173] Cyanobacterial-derived saxitoxins and microcystins are assessed using the enzyme linked immunosorbent assay (ELISA, Abraxis). Known cyanobacteria-derived biotoxins including microcystins, nodularin, cylindrospermopsin and anatoxin-a are screened using an Agilent 1260 UHPLC instrument equipped with a 1260 DAD detector capable of scanning the 200-800 nm range. Some target toxins may be monitored based on species already identified in the community (for example Phormidium sp.). Random sampling of the communities for this suite of common toxins may be identified to verify that unknown toxin-producers are not present in the tychoplankton. Lysinoalanine may be measured by GC-FID after derivatization, and compared with results of HPLC analysis with fluorescent detection of dabsyl derivatives.

[00174] Feed trials on fish with algae biomass and single-cell protein preparations.

[00175] Nutrient digestibility is determined by feeding the fish with a test diet, which include 30% of the test ingredient and 70% of reference diet (NRC, 2011). Fecal samples are collected and measured for the targeted nutrients. For feed trials, a series of test diets containing different levels of the algae ingredients is fed to juvenile Mozambique tilapia (Oreochromis mossambicus), which is one of the major representative species of fish in aquaculture. Three replications, for example, may be conducted per dietary treatment. At the end of 8-12 week of feeding, the survival, growth rate, feed conversion ratio, and estimate changes in production cost of tilapia fed with different diets are measured.

[00176] Determination of the quality and safety of fish fillet produced using algae- based feed.

[00177] The carcass of fish from each dietary treatment is sampled and used to determine the proximate composition (dry matter, ash, protein, lipid and gross energy). Sensory quality is evaluated (Erickson et al., 2007) for odor/smell, appearance (color), texture (firmness, fattiness and moistness), and flavor (sweetness, earthiness or off flavor). Accumulation of identified toxins is determined for tissues including liver, muscle and viscera.

[00178] Uses for the SAFR apparatuses, SAFR systems and SAFR methods

[00179] Producing low cost feeds by intensive aquaculture practices is one of the main priorities of modern agriculture. SAFR technology may be used to produce low cost biomass at prices competitive for feeds. The algae biomass produced using the methods disclosed herein may be used as a low-cost component for animal feeds (< $1 kg-1). Tychoplankton biomass produced by SAFR technology is an important protein source for animal feeds.

[00180] Water (AGM) consumption is very little using the SAFR method. Energy consumption is also very low, meaning that the SAFR technology may be supplied almost exclusively from solar energy. At the sustainability level, SAFR technology has the following performance characteristics per kg of algae biomass produced, expressed in dry weight: < $1 overall production cost; < 50 L water (AGM) consumption and < 5 kwh electricity consumption. These features are very important at the societal level, because SAFR algae technology is a good fit for expanding food production to arid areas (with low quality soil, warm and hot climate, low precipitation levels and good sun exposure) such as deserts.

[00181] The following examples are offered by way of illustration and not by way of limitation.

6. EXAMPLES

[00182] 6.1. Example 1: Algae Biomass Production by SAFR System

[00183] This example demonstrates the use of an SAFR system to produce algae biomass.

The SAFR system used was a medium-scale SAFR system as shown in FIG. 3. Either solar light or artificial light may be used for photosynthesis.

[00184] Basic principles and advantages of SAFR systems

[00185] The SAFR approach addresses some of the limitations of algae farming. These are: high harvesting costs, excessive water consumption, high energy consumption and waste of nutrients (Howell, 2010). Flocculation is often used to lower the cost of algae farming and may be done in many ways: altering the CO ₂ supply; decreasing the pH; foaming; sonication; charge neutralization with electricity or flocculants; and introducing microorganisms that induce flocculation (Riebesell, 1989; Salim et al., 2011; Guo et al., 2013; Liu et al., 2014). Such flocculation methods adds cost to algae farming, some types of industrial flocculation render water unusable for recirculation (and require expensive treatment), and in some algae growth systems, after biomass is harvested, remaining water is discarded along with unused nutrients.

[00186] Advantages of the SAFR system come from its basic principles:

[00187] (1) low to moderate concentration of nutrients;

[00188] (2) water (or AGM) recirculated through a large mineralization reservoir where no photosynthesis occurs;

[00189] (3) constantly filtering, mineralizing and recycling nutrients from the planktonic organic matter;

[00190] (4) culturing algae that are competitive in the presence of dissolved organic carbon; [00191] (5) culturing algae that have a natural tendency to aggregate, yet do not attach to substrates;

[00192] (6) culturing a consortium of algae rather than monospecies;

[00193] (7) all water reused; and

[00194] (8) adding algae growth medium (AGM) periodically to compensate for nutrients removed as harvested biomass.

[00195] The biomass yield of medium-scale SAFR systems, installed in Los Angeles area was a 11 kg of DW m ^"2 of AGR yr ^"1 and water consumption was ~ 67 L m ² AGR yr ^"1 . This was equivalent to ~ 6 L kg ^"1 biomass, which was < 1 % off the water needed to produce same amount of soy protein, and < 2% off the water needed to produce algae biomass for oil (i.e. ~ 470 L kg ^"1 of DW, assuming farmed algae with 75% oil per DW; Wigmosta et al., 2011 ; White, 2011). Electrical energy consumption was also low, ~ 5 MWh mt ^"1 of DW, equivalent to $0.55 kg ^"1 of DW biomass. Lab cultures of Scenedesmus, Pediastrum, Planctonema and

Monoraphidium (main players in the SAFR floe) were lab grown with high concentration of nutrients, ~ 180 ppm nitrate and ~ 161 ppm phosphate (Bischoff and Bold, 1963; Starr and Zeikus, 1993; Grewe and Markus, 2000; Lee et al., 2014). The SAFR floe grew in less concentrated nutrient solutions, < 80 ppm ammonium, < 5 ppm nitrate and < 5 ppm phosphate. A sufficient supply of nutrients remained available to the floe culture because water circulated through a large reservoir and additional nutrients were added as nutrients were removed.

[00196] Design and operation of SAFR systems

[00197] Lab-scale SAFR apparatuses and SAFR systems (FIG. 2) were used to study growth principles of tychoplankton in flow-through environments, to analyze how various algae species and consortia contribute to the formation of algae floe, and to produce inoculum for medium-scale SAFR systems. Artificial light was supplied for photosynthesis. These systems used square media bottles with 1-5 layers of 8-16 mm diameter transparent borosilicate glass beads as AGM flow disrupters. The surface area of the algae growth raceway was about 120 cm ² . Mineralization of recirculated water was done by aeration with a target Biological Oxygen Demand (BOD) < 1,000 ppm.

[00198] Flow rate of AGM and total fluid volume of AGM was controlled, allowing experiments with both flow velocity and residence time. The inventors have observed that > 10 cm s ^"1 washed out the loose floe and favors algae attached to surfaces, while at < 0.1 cm s ^"1 planktonic algae were not washed away efficiently. Hence, in one embodiment, an AGM flow rate in the range of about 0.1 cm s ^"1 - 10.0 cm s ^"1 (+ 0.05 cm s ^_1) is used. In another embodiment, an AGM flow rate in the range of about 0.1 cm s ^"1 - 5.0 cm s ^"1 (+ 0.05 cm s ^_1) is used. In another embodiment, an AGM flow rate in the range of about 5.0 cm s ^"1 - 10.0 cm s ^"1 (+ 0.05 cm s ^_1) is used. In another embodiment, an AGM flow rate that is equal to or greater than 0.1 cm s ^"1 and less than or equal to 10.0 cm s ^"1 is used. Also, if flow velocity at night is too low, a combination of high floe biomass density, warm water and insufficient O2 leads to death by anoxia.

[00199] In lab-scale SAFR systems UV-sterilization of recirculated water was used to help limit the phytoplankton. Average water flow velocity was kept at < 3 cm s ^"1 and each culture was transferred 2-3 times a week into new sterile bottles with clean glass beads. After each transfer, the algae floe was allowed to settle between the beads for approximately 1 hour. Then, water circulation was restored at 0.1 cm s ^"1 and the flow was increased at a rate of ~ 1 cm min ¹ to the desired value.

[00200] Medium-scale SAFR systems (FIG. 3) were used to grow biomass for animal feed trials, to study and improve energy and water sustainability and to analyze effects of scaling-up. Solar light or artificial light may be used for photosynthesis. These systems contained one mineralization tank with 1 ,000 L capacity and one or a sequence of two to six AGRs. Each raceway had a surface area of 1.6-2.8 m ² , is made of UV resistant acrylonitrile butadiene styrene (ABS), and contained one layer of polished pebbles (3-5 cm shortest cross section) used as a floc-accumulating habitat. Water (i.e., AGM) depth was adjustable

(commonly 5-7 cm), and water (i.e., AGM) flow velocity was also adjustable (commonly 0.1-3 cm s ^"1 ). At 1 cm s ^"1 the water turnover time in the system was about 7 hours. Aeration in the mineralization reservoir was done by purging air at 5-10 L min ^"1 . Continuous aeration at night helped avoid anoxia in the algae floe deposit.

[00201] The following conditions have generated algae biomass yields of ~ 30 g m ^~2 of AGR day ^"1 in medium-scale SAFR systems running in the Los Angeles area: ~ 1 cm s ^"1 flow velocity; ~ 25-80 ppm ammonium; ~ 0.5-5 ppm nitrate; < 0.07 ppm nitrite; ~ 1.2-5 ppm phosphate; larger than 16: 1 mole ratio between nitrogen and phosphorus; < 500 ppm BOD; < 1,000 ppm COD; 7.5-8.7 pH; 18-24 ^° C; > 30% 0 ₂ saturation at night; and 1-10 mS/cm conductivity. Harvesting was done when floe biomass abundance in the AGRs had reached about 2.7-3.0 g of DW L ^"1 (at approximately one week intervals). Harvesting at more frequent intervals increases production costs. Harvesting less often produced more biomass per harvest, but lowered the overall yield. For harvesting, the water flow was stopped and the upper water layer was decanted. The loose floe sediment was pressure-washed to produce an algae slurry with a 1-2% biomass (DW). Then the algae slurry was allowed to settle in the cold room for 6- 12 hours, the upper water layer (~ 80-90 %) was decanted and the floe -rich sediment was concentrated by continuous flow centrifugation into an algae cake with ~ 13-20% DW. The algae cake was either dried or further processed into single-cell protein.

[00202] Processing algae cake into single-cell protein

[00203] Green microalgae biomass is known to be a good feed amendment in various farmed animals including chicken (Ross and Dominy, 1990; Toyomizu et al., 2001; Moraine et al.,1979), carp (Moraine et al., 1979), swine (Becker 2004), Atlantic salmon (Kiron et al., 2012), whiteleg shrimp (Kiron et al., 2012) and tilapia (Attalla and Mikhail, 2008). Due to low digestibility, the upper concentration limits for unprocessed algae is below 15% in most feeds. Yet, up to 50% substitution of tilapia meal diets has been shown to work with dried

Scenedesmus spp. (Badwy et al., 2008), the dominant algae in SAFR technology. The principal reason for the low digestibility of microalgae is the cell wall, which keeps cells intact, lowers exposure to proteolytic enzymes and limits the availability of proteins (Nasseri et al., 2011). Other limitations include: excess nucleic acids, mainly RNA (releasing purines which upon dissimilation produce urea and uric acid), inhibitory byproducts generated during biomass processing (e.g., lysinoalanine), proteins becoming locked in complexes with oxidized polyphenols, algal toxins and heavy metals (Hedenskog and Morgen, 1973; Nasseri et al., 2011 ; Finlay and Kohler, 1979; Kightlinger et al. 2014). Furthermore, conventional conversion factor (N x 6.65) for crude protein overestimates the protein content of algae biomass because while algae biomass contains excess of non-proteic nitrogen as nucleic acids, cell walls nitrogen and amines (Hedenskog and Morgen, 1973; Nasseri et al 2011). For these reasons, processing microbial (including algae) biomass into single cell protein increases nutrient content and digestibility and decreases toxicity (Moraine et al., 1979; Mahasneh, 1997; Nasseri et al., 2011). Therefore, measurements of true proteins and amino acids are very important when using algae in feeds.

[00204] A protocol was developed to produce single-cell protein (modified after

Hedenskog and Morgen, 1973; Becker, 2004; Nasseri et al 2011) and it was found that postprocessing of the Scenedesmus community from SAFR systems increased efficiency in feed trials. This protocol is summarized in the Methodology section (below).

[00205] Algae cultures [00206] In certain embodiments, green algae that are used are Scenedesmus quadricauda and species of Pediastrum, Planctonema and Monoraphidium. Scenedesmus spp. are very nutritious in animal feeds (Ahlgren and Hyenstrand, 2003; Badwy et al., 2008; Skrivan et al., 2010), resilient to grazers (Mayeli et al., 2005), easy to isolate and maintain and can grow as unicells or coenobia, a phenotype important for tychoplankton and controllable via nutrient availability and growth rate (Healey et al., 1975; Rocha et al., 2015). Scenedesmus obliquus is particularly important for its ability to auto-flocculate (Salim et al., 2011). Scenedesmus quadricauda was often used in nutrient removal and fat production (Wong et al., 2015).

Dissolved Organic Carbon (DOC) is an important controller in communities of Scenedesmus, Pediastrum and Planctonema (Dasi et al. 1998; Toyub et al., 2008; Weckstrom et al., 2010). Scenedesmus algae (S. quadricauda included) are capable of mixotrophy and can grow in media with high organic load (Gantar et al., 1991 ; Kim et al., 2007; Xiao et al., 2011; Welter et al., 2013; Rocha et al., 2015). Attachment of Scenedesmus sp. to surfaces was studied by Chen et al. (2014) and selection/environmental control can be applied to favor self-aggregated vs, biofilm or planktonic phenotype. The sinking rate and effects of turbulence on Scenedesmus quadricauda have also been studied (Titman and Kilhman, 1976; Hondzo et al., 1998; Hondzo and Lyn, 1999). In toxic water, Scenedesmus sp. accumulate metals and toxins (Hardy et al., 1985), but this is not a problem in controlled cultures with low level of pollutants.

[00207] Species of Pediastrum, Planctonema and Monoraphidium are also facile to culture (Day and Brand, 2006; Bogen et al., 2013). Pediastrum species are non-motile coenobia with important role in producing floe assemblages and induce flocculation of other algae as well (Grewe and Markus, 2000; Weckstrom et al, 2010). Planctonema is common in meso- and hyper-eutrophic conditions and is a nutritional opportunist adapted to variable environments (Dasi et al. 1998; Naselli-Flores et al., 2002; Chen et al., 2003; Qin et al., 2007). Planctonema has a characteristic growth in filaments and increases floe cohesion. Monoraphidium is common in aggregates and shown to be important in producing specific fats (Bogen et al., 2013). Other auto-flocculating green algae that may be used is Ankistrodesmus falcatus, also easy to grow (Salim et al., 2011).

[00208] Preventing biotoxins in SAFR products

[00209] Because producing feed substitutes is a main target of SAFR technology, it is essential to avoid the buildup of biotoxins. Biotoxins include chemicals produced by living cells (e.g. phycotoxins and bacteriotoxins) or chemicals produced as biomass is processed into feeds (e.g. lysinoalanine). Many cyanobacteria produce powerful toxins, and wide diversity of cyanotoxins exists including: microcystins (Dawson, 1998; Blaha et al., 2009); nodularins (Wood et al., 2012); anatoxins; cylindrospermopsin; and saxitoxins (da Silva, 2011). Production of toxins rarely occurs in the Chlorophyceae genera (Chiang et al., 2004), which can be used in SAFR systems. Some marine species of diatoms produce the toxin domoic acid, but this chemical is not present in freshwater diatoms. In SAFR systems, biotoxins are most likely to be produced by uncontrolled cyanobacteria coupled with overcrowding, depletion of nitrogen and anoxia.

[00210] The green algae cultured in SAFR floes belonged to non-toxic genera,

Scenedesmus, Pediastrum, Planctonema and Monoraphidium. Very few cyanobacteria (< 0.1 % VohVol) were observed. Yet, because contamination is frequent in mid- and large-scale algae growth systems, management plans must be in place for containing the growth of toxic microorganisms. For example, frequent harvesting and continuous water circulation and aeration help avoid anoxia and buildup of excess of floe biomass, bacteria and DOC. The system has to be periodically cleaned, sterilized and re-inoculated with selected algae cultures that do not contain toxic cyanobacteria. The frequency of re-starting the system depends on the evolution of the algae community, chemistry and biotoxins. Yet, because re-starting the SAFR system is disruptive to biomass production, proactive management to avoid undesirable algae is preferable.

[00211] For example, increase in the N:P mole ratio above 16: 1, and providing nitrogen predominantly in mineral ionic forms (such as ammonium and nitrate) limits the growth of cyanobacteria for three reasons. First, cyanobacteria are better suited to nitrogen deprivation because they can fix N ₂ . Second, cyanobacteria are not well adapted to efficient use of ammonium and nitrate (Reuter et al., 1986). The third reason is allelopathic control with inhibitors of cyanobacteria such as gallic acid, indole, ethyl-2-methylacetoacetate and 3-oxo-a- ionone; some of these chemicals are produced by periphyton algae when total the dissolved phosphorus is low (Wu et at., 2010). Furthermore, adding sulfate to the AGM helps control cyanobacteria, because sulfate inhibits Mo-dependent N2-fixation (Marino et al., 2013).

Consequently, SAFR systems should not turn anoxic, in order to limit the reduction of dissimilatory sulfate with DOC to sulfide.

[00212] Sustainability features and advantages of SAFR apparatuses, SAFR systems, and SAFR methods [00213] Biomass yield is large (30 g m ^"2 d ^"1 ), comparable to other feed crops (soy and corn).

[00214] Water consumption is very low (~ 6 L kg ^"1 product DW).

[00215] Electrical energy consumption is low (~ 5 MWh mt ^"1 DW) and may be decreased even further.

[00216] More than 90% of the electrical energy needed can come from renewable sources (solar).

[00217] The cost of producing algae biomass is low (< $1 kg ^"1 ), within range of animal feeds ($1-2 kg ^"1 ).

[00218] SAFR systems can be easily automated.

[00219] Algae meal can help to lower the demand for fish meal and the pressure on ocean fishing for feed.

[00220] SAFR apparatuses, SAFR systems, and SAFR methods can find applications in water treatment, such as removal of nutrients (nitrogen and phosphorus) from waste water, eutrophic aquifers and aquaculture.

[00221] SAFR technology can become a farming method supporting the aquaculture of fish and shrimp in arid geographic areas where water is limited and the climate is hot (such as deserts).

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[00224] Algae biomass was grown according to the methods disclosed in Section 6.1 above. The content of a dried sample of the algae biomass was analyzed through the services of a commercial laboratory (Intertek Agricultural Services, New Orleans, LA 70122). The results of the analysis are set forth in Table 2 below. These results confirm that the sample had a high protein content of 36.52 wt% and therefore, that algae biomass is a promising high-quality aquafeed amendment. These results also confirm that algae biomass is rich in the specific amino acids listed in Table 2.

Table 2. Analysis of algae biomass

Test Method Result Units

Dry Matter Calculation from Moisture 96.57 wt%

Lipid (Crude Fat) AOCS Ba3-38 1.10 wt%

*Protein AOCS Ba4e-93 36.52 wt%

Crude Fiber AOCS Ba6-84 7.6 wt%

Ash AO AC 942.05 25.23 wt%

Acid Detergent Fiber AOAC 973.18 35.2 wt%

Calories Calculation 290.86 calories/ lOOg

Moisture AACC 44.15.02 3.43 wt%

*Acid Stable Amino Acids

Aspartic Acid 3.12 g/100g

Threonine 1.40 g/lOOg

Serine 1.33 g/lOOg

Glutamic Acid 3.29 g/lOOg

Proline 1.44 g/lOOg

Glycine 1.95 g/lOOg

Alanine 2.03 g/lOOg

Valine 1.70 g/lOOg

Methionine 0.450 g/lOOg

Isoleucine 1.08 g/lOOg

Tyrosine 0.910 g/lOOg Test Method Result Units

Phenylalanine 1.36 g/100g

Histidine 0.520 g/100g

Lysine 1.67 g/100g

Arginine 1.48 g/100g

*Sulfur Amino Acids (after

oxidation )

Cysteine 0.463 g/100g

Methionine 0.510 g/100g

^Tryptophan ( alkaline

hydrolysis)

Tryptophan 0.406 g/100g

Abbreviation: AOAC, AOAC International; AOCS, American Oil Chemists' Society; AACC, American Association for Clinical Chemistry

[00225] 6.3. Example 3: Feed trials on black soldier fly larvae (Hermetia illucens)

[00226] The effect of adding microalgae powder produced by the SAFR method to the diet of an insect, black soldier fly larvae (Hermetia illucens), was tested. In experiments, black soldier fly larvae were fed with a diet of 100 % beer mash (control), 25% beer mash and 75% microalgae powder, or 100% microalgae powder. A food conversion ratio was obtained ranging between 0.95 and 1.2 with an average of approximately 1. The larvae were fed for 10 days at approximately 25 C and harvested by separation from the growth mixture and washing on the twelfth day. The replacement of 25% beer mash with microalgae powder resulted in nonsignificant change in growth (based on triplicates and 1 standard deviation differences). The replacement of 50% of the beer mash with algae powder halted larval growth and feeding the larvae with 100% algae powder resulted in loss of weight (varying between 10 and 50% loss of weight in 10 days).

[00227] Samples of SAFR apparatuses, SAFR systems, and SAFR methods

[00228] Samples of apparatuses, systems and methods that are described herein are set forth in the following numbered paragraphs:

[00229] 1. An apparatus for growing algae biomass comprising: at least one Algae Growth Raceway (AGR);

an Algae Growth Medium (AGM) reservoir functionally connected to the AGR;

at least one AGM flow disrupter positioned in the AGR; and

an AGM circulation system for circulating AGM through the at least one AGR.

[00230] 2. The apparatus of paragraph no. 1, further comprising at least one filter or at least one filtration system disposed in a fluid flow path of the circulating AGM.

[00231] 3. The apparatus of paragraph no. 1, further comprising an aeration system.

[00232] 4. The apparatus of paragraph no. 1, wherein the aeration system is positioned in the AGM reservoir.

[00233] 5. The apparatus of paragraph no. 1, wherein the AGM circulation system collects and recirculates AGM.

[00234] 6. The apparatus of paragraph no. 1, further comprising a plurality of algae growth raceways (AGRs), wherein the AGRs are connected in sequence or in parallel.

[00235] 7. The apparatus of paragraph no. 1, wherein the Algae Growth Raceway (AGR) comprises:

an algae trough, wherein at least one algae growth medium (AGM) flow disrupter is disposed in the algae trough;

an inflow system;

an outflow (discharge) system; and

a lid for sealing the AGR.

[00236] 8. The apparatus of paragraph no. 1, wherein the AGR further comprises an algae collecting portion.

[00237] 9. The apparatus of paragraph no. 7, wherein the AGR comprises an algae collecting portion, and wherein the algae collecting portion is disposed on the surface of the algae trough to keep algae in place for growing.

[00238] 10. The apparatus of paragraph no. 7, wherein the algae trough comprises grooved portions, indentations or depressions in which the algae are retained and grow.

[00239] 11. The apparatus of paragraph no. 1, further comprising algae growth medium (AGM), wherein the AGM is stored in the AGM reservoir.

[00240] 12. The apparatus of paragraph no. 1, further comprising a water management system, wherein the water management system:

monitors AGM flow, monitors a parameter, wherein the parameter is an AGM chemistry parameter or an

environmental parameter, or

adjusts the parameter to a desired value.

[00241] 13. The apparatus of paragraph no. 12, wherein the AGM chemistry parameter or environmental parameter is pH, ammonium content, nitrate content, nitrite content, phosphate content, oxygen content, redox potential, temperature, light intensity, light periodicity, salinity, or AGM flow rate.

[00242] 14. The apparatus of paragraph no. 1, further comprising a sterilization system.

[00243] 15. The apparatus of paragraph no. 1, further comprising a mineralization system or mineralization tank.

[00244] 16. An Algae Growth Raceway (AGR) comprising:

an algae trough, wherein at least one algae growth medium (AGM) flow disrupter is disposed in the algae trough;

an inflow system;

an outflow system; and

a lid for sealing the AGR.

[00245] 17. The AGR of paragraph no. 16, having a regulated flow-rate of AGM.

[00246] 18. The AGR of paragraph no. 16, further comprising an algae collecting portion.

[00247] 19. The AGR of paragraph no. 18, wherein the algae collecting portion is disposed on the surface of the algae trough to keep algae in place for growing.

[00248] 20. The AGR of paragraph no. 18, wherein the algae collecting portion comprises grooved portions, indentations or depressions in which algae are retained and grow.

[00249] 21. A system for growing algae biomass comprising:

the apparatus of paragraph no. 1 ; and

algae growth medium (AGM).

[00250] 22. The system of paragraph no. 21, further comprising algae.

[00251] 23. The system of paragraph no. 22, wherein the algae comprises Scenedesmus spp.

(e.g., Scenedesmus quadricauda; S. acuminatus; S. spinosus), Pediastrum spp. (e.g., Pediastrum duplex; P. tetra), Planctonema spp., Monoraphidium spp. or Ankistrodesmus spp.

[00252] 24. The system of paragraph no. 21, wherein AGM is stored in the AGM reservoir and circulated in the apparatus by the AGM circulation system. [00253] 25. The system of paragraph no. 21, wherein the AGM comprises fresh water, salt water, seawater, waste water, gray water, recycled water, or eutrophic water.

[00254] 26. The system of paragraph no. 21, wherein the AGM consists of fresh water, salt water, seawater, waste water, gray water, recycled water, or eutrophic water.

[00255] 27. The system of paragraph no. 21, wherein AGM is supplemented with an AGM supplement.

[00256] 28. The system of paragraph no. 21, wherein the AGM supplement is compost tea, fermentation leachate, silage leachate, leachate from anaerobic digestion, leachate from decaying algae biomass, liquid distillation residue, beer-making liquid residue, food waste liquid residue, liquid ejecta of animal farming, microbial sludge, a liquid byproduct of an aquaculture facility, a mineral that supports or augments algae growth, a mineral blend that supports or augments algae growth, or a mineral solution that supports or augments algae growth, or a combination thereof.

[00257] 29. A method for growing algae biomass comprising:

providing the system of paragraph no. 21, wherein AGM is contained in or collected in the AGM reservoir of the apparatus;

providing an algae culture;

flowing AGM from the AGM reservoir to the AGR of the apparatus;

introducing the algae culture into the AGM;

illuminating the system with natural or artificial light; and

growing algae from the algae culture to produce algae biomass.

[00258] 30. The method of paragraph no. 29, wherein AGM is recirculated back to the AGM reservoir from the AGR.

[00259] 31. The method of paragraph no. 29, further comprising harvesting algae biomass from the system.

[00260] 32. The method of paragraph no. 29, wherein the harvesting comprises:

draining the AGM from the AGR in the system; and

washing the algae biomass from the system.

[00261] 33. The method of paragraph no. 29, further comprising collecting or removing the AGM from the apparatus.

[00262] 34. The method of paragraph no. 29, wherein the collecting or removing comprises pumping the AGM from the apparatus. [00263] 35. A method for remediating an aqueous solution is provided comprising:

providing the apparatus of paragraph no. 1 ;

providing an aqueous solution to be remediated, wherein the aqueous solution is an algae growth medium (AGM);

growing algae in the apparatus, and

separating the aqueous solution from algae in the apparatus.

[00264] 36. The method for remediating of paragraph no. 35, further comprising collecting or removing the aqueous solution from the apparatus.

[00265] 37. The method for remediating of paragraph no. 36, wherein the collecting or removing comprises pumping the aqueous solution from the apparatus.

[00266] 38. The method for remediating of paragraph no. 35, wherein algae is already present or growing in the aqueous solution to be remediated.

[00267] 39. The method for remediating of paragraph no. 35, further comprising recovering algae biomass from the apparatus.

[00268] 40. The method for remediating of paragraph no. 35, further comprising growing a desired species of algae in the aqueous solution in the apparatus.

[00269] It will be appreciated that variants of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems, methods or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

[00270] While embodiments of the present disclosure have been particularly shown and described with reference to certain examples and features, it will be understood by one skilled in the art that various changes in detail may be effected therein without departing from the spirit and scope of the present disclosure as defined by claims that can be supported by the written description and drawings. Further, where exemplary embodiments are described with reference to a certain number of elements it will be understood that the exemplary embodiments can be practiced utilizing either less than or more than the certain number of elements.

[00271] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. [00272] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.