U.S. patent application number 13/773304 was filed with the patent office on 2013-10-17 for compositions and methods for continuous harvesting of suspension growth cultures.
This patent application is currently assigned to SOLIX BIOFUELS, INC. The applicant listed for this patent is SOLIX BIOFUELS, INC. Invention is credited to Richard CROWELL, Tianxi ZHANG.
Application Number | 20130273630 13/773304 |
Document ID | / |
Family ID | 44816126 |
Filed Date | 2013-10-17 |
United States Patent
Application |
20130273630 |
Kind Code |
A1 |
ZHANG; Tianxi ; et
al. |
October 17, 2013 |
COMPOSITIONS AND METHODS FOR CONTINUOUS HARVESTING OF SUSPENSION
GROWTH CULTURES
Abstract
Embodiments herein concern compositions, methods and uses for
harvesting suspension cultures or decontaminating waters. In
certain embodiments, suspension microorganism cultures can comprise
algal cultures. In some embodiments, harvesting suspension cultures
may include using a composition capable of interacting with the
culture in order to separate the culture from a liquid or
media.
Inventors: |
ZHANG; Tianxi; (Fort
Collins, CO) ; CROWELL; Richard; (Fort Collins,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SOLIX BIOFUELS, INC; |
|
|
US |
|
|
Assignee: |
SOLIX BIOFUELS, INC
Fort Collins
CO
|
Family ID: |
44816126 |
Appl. No.: |
13/773304 |
Filed: |
February 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13072527 |
Mar 25, 2011 |
8399239 |
|
|
13773304 |
|
|
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|
61317863 |
Mar 26, 2010 |
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Current U.S.
Class: |
435/173.9 ;
210/702; 435/257.1 |
Current CPC
Class: |
C02F 2303/20 20130101;
C12N 13/00 20130101; C02F 1/488 20130101; C02F 1/5236 20130101;
C12M 47/02 20130101; C02F 1/66 20130101; Y02W 10/37 20150501; C02F
2303/04 20130101 |
Class at
Publication: |
435/173.9 ;
435/257.1; 210/702 |
International
Class: |
C12N 13/00 20060101
C12N013/00 |
Claims
1. A method for harvesting a suspension of an organism in a liquid,
comprising: adding a magnesium agent to the suspension to produce a
flocculant comprising the organism and the magnesium agent;
separating the flocculant from the liquid; and harvesting the
organism.
2. The method of claim 1, wherein the flocculant is produced at a
pH about 9.0 to pH about 11.5.
3. The method of claim 1, wherein the magnesium agent is a
positively-charged agent comprising a magnesium hydroxide.
4. The method of claim 1, wherein the separating step comprises
adding a negatively charged magnetic particle and exposing the
flocculant to a magnetic field.
5. The method of claim 1, wherein harvesting the organism occurs
after the flocculant is exposed to a reduced pH of about 6.0 to
about 7.5.
6. The method of claim 1, further comprising: adding a negatively
charged non-magnetic material to the suspension to produce a
complex comprising the flocculant and the negatively charged
non-magnetic material; and allowing the complex to settle out of
the suspension.
7. The method of claim 6, wherein the negatively charged
non-magnetic material is selected from silica, tungsten, aluminum,
aluminum hydroxides, copper, allay, rare earth materials, ceramic
materials, clay, glass, calcium, and carbon, organic materials and
composite materials.
8. The method of claim 6, wherein the negatively charged
non-magnetic material has a particle size of 0.5 .mu.m to 10
.mu.m.
9. The method of claim 6, further comprising harvesting the
non-magnetic material for reuse.
10. The method of claim 1, wherein the organism is an algae.
11. The method of claim 10, wherein the algae is further harvested
for an algal yield.
12. The method of claim 11, wherein the algae is further harvested
for biofuel extraction.
13. A method for decontaminating water comprising a particulate or
contaminant, the method comprising: adding a positively charged
magnesium agent to the water to form a flocculant comprising the
positively charged magnesium agent and the particulate or
contaminant; adding a non-magnetic material to the contaminated
water to produce a complex comprising the particulate or
contaminant and the flocculant; allowing the complex to settle out
of the water; separating the complex from the water to produce
decontaminated water; and reusing the decontaminated water.
14. The method of claim 13, wherein the decontaminated water can be
used for non-potable or potable use.
15. A method for separating a negatively charged particulate from a
liquid having a high concentration of magnesium ions, the method
comprising: increasing the pH of the liquid to a pH of at least 8.5
to about 12.5 to produce a first complex comprising the particulate
and a magnesium precipitate; adding a negatively charged inorganic
particle to the liquid to form second complex comprising the first
complex and the negatively charged inorganic particle; and
separating the second complex from the liquid.
16. The method of claim 15, further comprising exposing the second
complex to a is exposed to a pH of about 6.0 to about 7.5 to
recover the negatively charged inorganic particle.
17. The method of claim 15, wherein the negatively charged
particulate is algae.
18. The method of claim 15, wherein the liquid contains from about
600 mg/L to about 2000 mg/L of magnesium in the form of an
inorganic salt.
19. The method of claim 15, wherein the negatively charged
inorganic particle has a density of from about 2.6 g/cm.sup.3 to
about 19.3 g/cm.sup.3.
20. The method of claim 15, wherein the negatively charged particle
has a particle size of from about 1 .mu.m to about 100 .mu.m.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit under 35 U.S.C.
119(e) of U.S. patent application Ser. No. 13/072,527 filed on Mar.
25, 2011 which claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/317,863 filed on Mar. 26, 2010, both of
which are incorporated herein by reference in their entirety for
all purposes.
FIELD OF THE INVENTION
[0002] Embodiments of the present invention generally report
methods and compositions for suspension cultures or waste waters.
In certain embodiments, compositions and methods concern separating
algae from a media or liquid. Other embodiments concern
compositions, methods and uses of a harvesting system or harvesting
agent for removing media compositions from suspension cultures and
reusing the harvesting agent. Yet other embodiments may concern
systems and methods for separating biomass from algae media for use
in biofuels production and generation of related algal
products.
BACKGROUND
Chemical Coagulation and Flocculation for Algae Harvesting
[0003] Algae harvesting can be challenging for algae biofuels
production due in part to few cost-effective technologies
available. One current technology is chemical coagulation and
flocculation, widely applied in water and wastewater treatment.
Coagulants typically function as neutralization of surface charges
of suspended particles in water. Neutralized particles are
typically still suspended in water as the coagulated particles do
not aggregate together to form big flocs. Thus, polymer flocculants
are added to bridge the neutralized particles for formation of big
flocs to permit settling out of the coagulates. Some polymer
flocculants are expensive even at low dose for algae
harvesting.
SUMMARY
[0004] Embodiments of the present invention generally report
methods and compositions for suspension cultures. In certain
embodiments, compositions and methods concern separating suspension
cultures (e.g. algae) from a media. Other embodiments concern
compositions, methods and uses of a harvesting system for removing
media compositions from suspension cultures. Yet other embodiments
may concern systems and methods for separating biomass from algae
media for use in biofuel production and generation of related algal
products. Some embodiments concern suspension cultures or
wastewaters including, but not limited to algae, bacteria, yeast,
fungi, suspended solids in water and wastewater particulates.
[0005] Certain embodiments of the present invention report magnetic
flocculation for harvesting a suspension culture or
particulate/microorganism removal from waters (e.g. wastewater). In
accordance with these embodiments, a culture may be an algal
culture. For example, algae can be adsorbed on surfaces of
magnetite particles, forming magnetically-linked algae complexes
capable of removal from growth media. In certain embodiments, the
magnetically-linked algae may be separated from a media using a
magnetic separator or sedimentation, such as by gravity or magnetic
field. In certain embodiments, algae and magnetite particles have a
negative charge in some media, which can result in an electrostatic
repulsion between them. Algae adsorption on magnetite particles
should have an attractive interaction to occur. Either algae or
magnetite can be changed into positively charged elements.
[0006] In certain embodiments, media for algae (e.g. for
Nannochloropsis oculata, Nannochloropsis salina or other algae)
cultivation can have high concentrations of magnesium ions (e.g.
600 mg/L, 1,000 mg/L or 2,000 mg/L or other) from inorganic salts
for the algal growth. Precipitation of magnesium hydroxides can
occur at high pH (e.g. about 8.5 to about 11.5, about 9.0 to about
11.0, about 9.5 to about 10.5) in media. Other solution pHs are
contemplated for use in precipitation of magnesium hydroxides. In
some embodiments, positively charged magnesium hydroxide
precipitates in suspension media can bind with both negatively
charged algae and magnetite based on electrostatic attraction to
form particle-algae complexes. In accordance with these
embodiments, particle-algae complexes can be captured by a magnetic
field (e.g. a magnet), concentrating the algae and separating the
algae from media or using other methods such as gravity. In certain
embodiments, magnesium hydroxides can flocculate algae at a high pH
(e.g. about 8.5 to about 11.5, about 9.0 to about 11.0, about 9.5
to about 10.5). Magnetite particles can tag algae to provide a
magnetic property, resulting in algal movement/attraction under a
magnetic field. In addition, particle-algae complexes can have a
higher density than non-particle algae. For example, magnetite
density can be about 5.0 g/cm.sup.3 to about 5.5 g/cm.sup.3. In
certain embodiments, some heavier flocculated algae can settle out
of solution by gravity. This process can produce less sludge in the
flocculation process.
[0007] In certain embodiments, agents used for flocculation or
coagulation can include, but are not limited to, iron oxides (e.g.
magnetite (Fe.sub.3O.sub.4), maghemite (Fe.sub.2O.sub.3),
FeOFe.sub.2O.sub.3,)) iron, steel, silica (sand), tungsten,
magnesium (e.g. magnesium chloride, magnesium hydroxides,
seawater), base (e.g. sodium hydroxides, lime), acid (e.g.
hydrochloric acid). In some embodiments, organic materials of use
in certain methods described herein can include, but are not
limited to, fiber, starch, wood, or polymers. In other embodiments,
composite materials can include, but are not limited to,
carbon-fiber, glass-plastics, silica-polymers, metal-polymers,
ceramic-polymers, and clay-polymers. In yet other embodiments,
agents of use to modify pH can include, but are not limited to,
chemical agent, a gas (e.g. air for pH increase, CO.sub.2 for pH
decrease), or other suitable agent capable of modifying pH of a
suspension culture in order to facilitate flocculation of the
culture or other matter. Some embodiments can include agents
capable of easy manipulation or that are easy to eliminate from the
suspension as necessary.
[0008] In certain methods disclosed herein, magnetic flocculation
can be a simple and efficient method to separate algae (e.g.
Nannochloropsis oculata and Nannochloropsis salina) from media. In
one embodiment a suspension culture can be tagged with a magnetic
or heavy material. The tagging magnetic or heavy material can
include, but are not limited to, iron oxide, tungsten, silicon,
magnetic material aluminum hydroxide, iron, iron sulfate, sand or
other suitable heavy material. In accordance with these
embodiments, the tagging or heavy material can be added at a basic
pH where precipitates can be formed. A basic pH contemplated for
some embodiments herein can include a pH of about 8.5 to about
11.5, about 9.0 to about 11.0, about 9.5 to about 10.5.
[0009] In one embodiment, algae can be tagged with iron oxide
particles at a basic pH to form magnesium hydroxide precipitates.
In certain embodiments, pH of a media or solution can have an
effect on harvesting performance. For example, algae can be
harvested (e.g. about 97%) through the enhanced settling by a
magnetic separator. In accordance with this example, about 90% of
media could be removed by gravity sedimentation of the magnetic
algae. In other embodiments, suspension cultures precipitated or
drawn to a magnetic separator may be concentrated. Some examples
concern concentrating algae associated with magnetic particles. In
other embodiments, compositions and methods can be used to separate
cultures from the magnetic or heavy particles using a gradual or
sharp adjustment in pH. In certain embodiments, the pH may be
decreased to pH of about 6.0 to a pH of about 7.5. In addition,
materials of use to precipitate and/or concentrate suspension
cultures may be recycled for reuse.
[0010] In other embodiments, magnetic flocculation for harvesting a
microorganism contemplated herein can be performed without base
addition and adjustment of the pH. In other embodiments, agents
used for flocculation or coagulation, iron oxides (e.g. magnetite
(Fe.sub.3O.sub.4), maghemite (Fe.sub.2O.sub.3), FeO
Fe.sub.2O.sub.3) iron, steel, silica (sand), tungsten, magnesium
(e.g. magnesium chloride, magnesium hydroxides, seawater), base
(e.g. sodium hydroxides, lime), acid (e.g. hydrochloric acid) can
be added to a culture from about 500 mg/L to about 2500 mg/L and pH
can be adjusted as necessary to induce flocculation. In one
embodiment, magnesium can be added to a culture at about 2,000 mg/L
and pH adjusted to about 9.5 wherein subsequent algae harvesting
can be about 99% recovery of the algae.
[0011] In certain embodiments, compositions contemplated herein may
concern suspension cultures or wastewaters in combination with
magnetic or heavy particles at a basic pH for
precipitation/concentration followed by a more neutral or slightly
acidic pH allowing separation of the cultures.
[0012] In some embodiments, magnetic or heavy particles may be
collected and regenerated for use in another suspension culture or
waste water. In other embodiments, continuous culturing and
concentrating techniques disclosed herein may be used for cost
effective and rapid suspension culture harvesting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] In the following sections, various exemplary compositions
and methods are described in order to detail various embodiments.
It will be obvious to one skilled in the art that practicing the
various embodiments does not require the employment of all or even
some of the details outlined herein, but rather that
concentrations, times and other details may be modified through
routine experimentation. In some cases, well-known methods or
components have not been included in the description.
[0014] FIG. 1 represents a schematic diagram of the magnetic
flocculation where A represents negatively charged organisms;
+represents positively charged precipitates; M represents
negatively charged magnetite and PM represents a permanent magnet.
The flow diagram represents complex formation and magnetic
attraction of the complex.
[0015] FIGS. 2A-2D represents microscopic images of magnetite
dispersion in suspension cultures at basic pH in various
concentrations of iron oxides 2.0% (A); 5.0% (B), 7.6% (C) and
10.3% (D).
[0016] FIGS. 3A-3C represents images of magnetic separation of
algae from a solution before magnetic application (A); during early
magnetic field exposure (B); and magnetically concentrated
complexes of algae (C).
[0017] FIGS. 4A-4B represents microscopic images of raw algae
before (A) and after flocculation (B).
[0018] FIG. 5 represents a schematic of a procedure using a dye
indicator during a flocculation process where an algae culture is
centrifuged, then the pH is adjusted to permit precipitation then a
dye is added that associates with the precipitates as an
indicator.
[0019] FIGS. 6A-6B illustrate photos of dye indicator in a sample
for formation of positive charged precipitates as illustrated in
FIG. 5.
[0020] FIG. 7 represents effects of solution pH on algae
harvesting.
[0021] FIG. 8 represents effects of solution pH as a function of
NaOH concentration.
[0022] FIG. 9 represents an exemplary plot of solution pH as a
function of Ca(OH).sub.2 concentration.
[0023] FIG. 10 represents an exemplary plot of solution pH as a
function of NH.sub.4OH concentration.
[0024] FIGS. 11A and 11B represent effect of magnetite content on
algae harvesting, (A) and (B) represent percent algae
harvested.
[0025] FIG. 12 illustrates recovered magnetite at a reduced pH.
[0026] FIGS. 13A-13B represents microscopic images of before (A)
and after the magnetite removal (B).
[0027] FIG. 14 represents an exemplary experimental protocol of
regeneration and reuse of magnetite particles where a culture is
mixed at a basic pH with a magnetite, vortexed, decant the
supernatant to leave the paste and subsequently separating the
magnetite from the concentrated algae.
[0028] FIG. 15 illustrates a bar graph representing harvesting
suspension cultures with reusable magnetite.
[0029] FIG. 16 represents effects of magnetite contents on the
algae sedimentation by gravity.
[0030] FIG. 17 represents sedimentation performance of suspension
cultures using tungsten.
[0031] FIG. 18 represents a comparison of magnetite and tungsten on
sedimentation by gravity of a suspension culture.
[0032] FIG. 19 represents experimental protocols of magnetic
harvesting in presence of magnetite where a culture is mixed at a
basic pH with a magnetite, mixed, separating the magnetite from the
concentrated algae and settling by gravity.
[0033] FIG. 20 represents photographs of enhancement of suspension
culture settling by a magnetic field for 1.5 to 31 minutes after
settling.
[0034] FIGS. 21A-21D represents microscopic images of suspension
cultures during the harvesting process, A. algae at 10 and 40 times
magnification, B. separator influent at 10; 40 times magnification;
C. effluent before settling and D. effluent after settling.
[0035] FIG. 22 represents a schematic protocol of suspension
culture separation using mixing/settling tank #1 and
mixing/settling tank #2 where algae broth is separated into
concentrated algae and a magnetic slurry for reuse.
[0036] FIG. 23 represents a histogram of algae biomass using flow
cytometry analysis.
[0037] FIG. 24 represents harvesting of a culture using an
exemplary apparatus disclosed herein.
[0038] FIGS. 25A and 25B are photographs illustrating the lack of
certain particles present after magnetic separation.
DEFINITIONS
[0039] As used herein, "a" or "an" may mean one or more than one of
an item.
[0040] As used herein, "about" may mean up to and including plus or
minus five percent, for example, about 100 may mean 95 and up to
105.
[0041] As used herein, "tag," "tagging" or "tagged" may mean
attaching a particle or agent to a microorganism. For example, a
tagged alga can be algae attached to a particle for harvesting or
other purpose as described herein.
DETAILED DESCRIPTION
[0042] In the following sections, various exemplary compositions
and methods are described in order to detail various embodiments.
It will be obvious to one skilled in the art that practicing the
various embodiments does not require the employment of all or even
some of the details outlined herein, but rather that
concentrations, times and other specific details may be modified
through routine experimentation. In some cases, well-known methods
or components have not been included in the description.
[0043] Embodiments herein represent methods, and compositions for
harvesting and using harvest suspension culture yields. In some
embodiments, the cultures can be algae, bacteria, fungi or yeast
cultures. Products contemplated herein for production from the
algal yields can include, but are not limited to, biofuels,
protein, vitamins, carbohydrates and/or amino acids.
[0044] In certain embodiments, particles or heavy materials may be
used as coagulants or concentrators of suspension cultures or
wastewaters (e.g. for removal of particulates or microorganisms).
For example, in some embodiments, microorganisms can associate with
particles or heavy materials by adjusting the condition of the
culture media in order to harvest the microorganisms.
[0045] Previously, it was demonstrated that adsorbed algae
particles can be removed from water by a magnetic field. Algae were
removed by passing through a high gradient magnetic filtration
(HGMF). In this study about 90% of algae were removed at 500-1,200
mg/L of magnetite as magnetic seeds in laboratory batch
experiments. One of the problems of this method was that removal of
the algae required a large concentration of iron ions as a primary
coagulant and iron coagulants tended to modify the algae surfaces
reducing production of the algae and the coagulants could not be
recycled for reuse making it an expensive process.
[0046] In some embodiments, algae surfaces can be modified prior to
adsorption using methods disclosed herein. In certain embodiments,
algal suspension cultures can be changed to hydrophobic cultures
prior to harvesting. In accordance with these embodiments,
magnetite particles often do not meet such a hydrophobic
requirement for association, so magnetite particles can be modified
to be hydrophobic using, for example, a silanization reaction.
Algal cells can be adsorbed on the magnetite surfaces and
algae-magnetite complex particles can be removed from the solution
using for example, a magnet. Thus, algae can be harvested from any
solution, for example, a media or other liquid. However, this
method requires the silanized hydrophobic magnetite, which leads to
an expensive process. In addition, treated algae also should be
hydrophobic surfaces, which might not be achieved for many species
of algae.
[0047] Some embodiments of the present invention concern algae
harvesting using flocculation or coagulation techniques. In certain
embodiments, methods disclosed herein may concern magnesium
hydroxide flocculation. In accordance with these embodiments, when
magnesium ions are present in a solution, an increase in pH can
lead to precipitation of magnesium hydroxides [Mg(OH).sub.2] shown
in the equation (1) below, previously submitted.
Mg.sup.2++2OH.sup.-=Mg(OH).sub.2.dwnarw. (1)
[0048] Solution pH plays a key role in magnesium hydroxide
precipitation. The figure below represents magnesium hydroxide
precipitation as a function of pH. The precipitation starts at
approximately pH 9.5 and completes at approximately pH 11.5. In
certain embodiments, an elevated pH may be about 9.0 to about 12.0,
or about pH 9.5 to about 11.5. Magnesium hydroxides are gelatinous
precipitates that carry positive charges. The mechanism of
magnesium hydroxide flocculation is at least in part an
electrostatic bridging where positively charged magnesium hydroxide
precipitates flocculate negative charged particles.
[0049] In certain embodiments, algal flocculation methods and
compositions were used in the presence of magnesium at basic pH
levels as indicated above.
[0050] It has been demonstrated that algal flocculation occurs at
high pH (e.g. about 9.0 to about 11.5). Algal flocculation can
occur by adding seawater in concentration of about 5-10% (v/v).
Both negatively charged calcium carbonate (CaCO.sub.3) and
positively charged Mg(OH).sub.2 could be precipitated at higher pH
(e.g. 10.2) when lime was added. A continuous fluidized reactor was
designed and tested. High suspended solid removal (e.g. 95%) was
reportedly obtained at pH values of 11.8-12.0. One of the problems
with these approaches was large amounts of sludge, however, were
generated in use of magnesium hydroxide precipitates, which can
hinder adoption of this process for commercialization application.
Gelatinous precipitates of magnesium hydroxides might lead to
increased formation of sludge in a loose structure. Therefore,
eliminating or reducing the sludge problem was a follow-on
issue.
[0051] Embodiments herein present solutions to alleviate or reduce
generation of sludge or other issues faced by some of these
previously disclosed methods. In some embodiments herein, algae can
be separated by using magnetic separation technologies followed by,
for example, changes in compositions in order to harvest the
cultures. In some embodiments, solution pH may be adjusted in order
to facilitate interaction between a culture (or contaminant) and
magnetite (e.g. using a base, such as sodium hydroxide). Magnetite
(Fe.sub.3O.sub.4) particles at different concentrations and
particle sizes can be added to algae cultures. Subsequently, a
permanent magnet can be applied to remove flocculated algae. Then,
the magnetite can be recovered, by a solution pH decrease, by
addition of for example, an acid (e.g. hydrochloric acid) where the
magnesium hydroxide precipitates were dissolved in solution,
following that an electrostatic repulsion between the algae and the
magnetite occurred. Concentrated algae can be obtained by decanting
supernatant when applying a magnetic field.
[0052] In some embodiments, a suspension culture can include, but
is not limited to, algae, bacteria, yeast, and fungi. Other
embodiments can include removal of suspended solids or
microorganisms in water and wastewater clean-up or contaminant
removal.
[0053] Algal strains contemplated for harvesting or concentration
herein can include, but are not limited to, Phaeodactulum
tricornutum, Chlorella protothecoides, Nannochloropsis salina,
Nannochloropsis sp, Tetraselmis succica, Tetraselmis chuii,
Botrycoccus braunii, Chlorella sp., Chlorella ellipsoidea,
Chlorella emersonii, Chlorella minutissima, Chlorella salina,
Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella
sorokiniana, Chlorella vulgaris, Chroomonas salina, Cyclotella
cryptica, Cyclotella sp., Dunaliella salina, Dunaliella bardawil,
Dunaliella tertiolecta, Euglena gracilis, Gymnodinium nelsoni,
Haematococcus pluvialis, Isochrysis galbana, Monoraphidium minutum,
Monoraphidium sp., Neochloris oleoabundans, Nitzschia laevis,
Onoraphidium sp., Pavlova lutheri, Phaeodactylum tricornutum,
Porphyridium cruentum, Scenedesmus obliquuus, Scenedesmus
quadricaula Scenedesmus sp., Stichococcus bacillaris, Spirulina
platensis, Thalassiosira sp. or combinations thereof. In other
embodiments, methods and compositions disclosed herein may be used
to harvest cyanobacteria or other suspension prokaryotic or
eukaryotic cultures.
[0054] Agents capable of coagulating or of use as a flocculant
include, but are not limited to, iron oxides (e.g. magnetite
(Fe.sub.3O.sub.4), maghemite (Fe.sub.2O.sub.3)), iron, steel,
silica (sand), tungsten, and magnesium agents (e.g. magnesium
chloride, magnesium hydroxides, seawater).
[0055] Solutions of use to modulate pH of a composition can include
base (e.g. sodium hydroxides, lime), acid (e.g. hydrochloric acid),
gas (e.g. air for pH increase, CO.sub.2 for pH decrease) or other
suitable agent.
[0056] In certain embodiments, media for algae (e.g. for
Nannochloropsis oculata, Nannochloropsis salina or other algae)
cultivation can have high concentrations of magnesium ions (e.g.
600 mg/L, 1,000 mg/L or 2,000 mg/L or other) from inorganic salts
for the algal growth. Precipitation of magnesium hydroxides can
occur at high pH (e.g. about 8.5 to about 11.5, about 9.0 to about
11.0, about 9.5 to about 10.5) in media. Other solution pHs are
contemplated for use in precipitation of magnesium hydroxides. In
some embodiments, positively charged magnesium hydroxide
precipitates in suspension media can bind with both negatively
charged algae and magnetite based on electrostatic attraction to
form particle-algae complexes. In accordance with these
embodiments, particle-algae complexes can be captured by a magnetic
field (e.g. a magnet), concentrating the algae and separating the
algae from media or using other methods such as gravity. In certain
embodiments, magnesium hydroxides can flocculate algae at a high pH
(e.g. about 8.5 to about 11.5, about 9.0 to about 11.0, about 9.5
to about 10.5). Magnetite particles can tag algae to provide a
magnetic property, resulting in algal movement/attraction under a
magnetic field. In addition, particle-algae complexes can have a
higher density than non-particle algae. For example, magnetite
density can be about 5.0 g/cm.sup.3 to about 5.5 g/cm.sup.3. In
certain embodiments, some heavier flocculated algae can settle out
of solution by gravity with or without magnetic flocculation. This
process can produce less sludge in the flocculation process.
Removal of Magnetite Residues from Settled Supernatant
[0057] Some embodiments concern methods for recycling and reusing
media. Magnetite residues in supernatant were observed after
gravity settling under a microscope. Magnesium hydroxide
precipitates might be one of the negative factors on the magnetite
settling in terms of water removal. Using pH adjustment, for
example, particles can be recovered for reuse for example using
magnetic capture. In addition, a high gradient magnet filter can be
used for scale-up operation and recovery of reusable materials. For
example, culture media can be recycled for algae cultivation after
the magnetite residues are removed.
Kits
[0058] In still further embodiments, kits are contemplated herein.
In some embodiments, a kit may include one or more composition
and/or concentrator for coagulating a suspension culture. Kits may
also include one or more suitable container means, magnetic
separating device, fluorescent dyes, pH adjusting agents, one or
more flocculant, one or more base solution, one or more other
extraction or harvesting agents.
EXAMPLES
[0059] The following examples are included to demonstrate certain
embodiments presented herein. It should be appreciated by those of
skill in the art that the techniques disclosed in the Examples
which follow represent techniques discovered to function well in
the practices disclosed herein, and thus can be considered to
constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope herein
Example 1
Feasibility of Magnetic Flocculation for Algae Harvesting
Dispersion of Magnetite Particles in Algae Broth
[0060] In one example, algae are attached to magnetite particles
for magnetic separation. In order to study the attachment, a light
microscope was used to observe the magnetite dispersion in the
algal growth media. One hypothesis is that positively charged
magnesium hydroxide precipitates could bind with both negatively
charged algae and magnetite based on electrostatic attraction. The
resulting particles can be captured by a permanent magnet, and then
algae are concentrated and separated from the media. FIG. 1
illustrates a possible mechanism of the magnetic flocculation
proposed. Magnesium hydroxides flocculate algae at a high pH.
Magnetite particles tag algae to generate a magnetic property,
where the algae can be manipulated in suspension or otherwise under
a magnetic field.
[0061] FIG. 2 illustrates the microscopic images of magnetite
dispersion in the algae broth in absence of a magnet. Magnetite
particles were attached with flocculated algae at pH 10.3 observed
in these images. Magnetite contents increased from 2.0% (w/v) (FIG.
2A) through 10.3% (w/v) (FIG. 2D). It is observed that more
magnetite particles continued to associate with the flocculated
algae with increasing magnetite contents from FIG. 2A to FIG. 2D.
These observations verified that the algae were capable of being
tagged with magnetic particles, indicating that the algal surfaces
were modified by magnetic particles. The magnetic algae could be
physically moved under a magnetic field. This magnetic modification
provides one basis of magnetic separation of use in algae
harvesting.
Algae Harvesting Using Magnetic Separation
[0062] FIGS. 3A-3C illustrates photos of three samples with a
permanent magnet applied. FIG. 3A demonstrates the raw algal broth
at pH 7.4 in absence of magnetite. There is no significant change
in this raw sample as magnesium hydroxide precipitates are not
formed at this pH. After increasing pH to 10.4 in FIG. 3B, the
sample turned into somewhat turbid, suggesting formation of
magnesium hydroxide precipitates. In this example, the magnet did
not have a significant influence on this sample likely due to
absence of magnetite particles. In FIG. 3C, most of green algae
with magnetite were captured by the magnet in presence of the
magnetite. Green color in solution was almost disappeared as algae
were moved on the tube wall with the magnetite. The supernatant can
be easily decanted when holding the magnet, resulting in the
concentrated algae obtained. This result demonstrated that the
algae were tagged with the magnetite and then were able to be
harvested by the magnet.
[0063] It is noted that the formation of positively charged
magnesium hydroxide precipitates may be required in certain
magnetic harvesting methods for algae. Both algae and magnetite are
associated with the magnesium hydroxides based on electrostatic
attraction, confirming the previous hypothesis. See FIGS.
3A-3C.
[0064] (a) Algae broth (pH 7.4) in absence of magnetite
[0065] (b) Algae broth (pH 10.4) in absence of magnetite
[0066] (c) Algae broth (pH 10.4) in presence of magnetite (1.8%
w/v)
Verification of Formation of Magnesium Hydroxide Precipitates
[0067] Algae were flocculated at pH 10.4 shown in FIG. 2 above.
FIGS. 4A and 4B represent the microscopic images of raw algae
before and after flocculation. The algae did flocculate at pH 10.4
(FIG. 4B), comparing with separated algal cells at pH 7.7 (FIG.
4A). (A) represents Raw Algae (pH 7.7); (B) Raw Algae (pH
10.4).
[0068] In order to further confirm the formation of positive
charged precipitates that activates the flocculation, a method was
developed using an anionic dye as an indication. A procedure of
this dye indicator is presented as a schematic in FIG. 5. At first,
raw algae were removed by centrifugation at initial pH 7.4 where
magnesium ions are still dissolved in the supernatant. The pH of
supernatant was increased to a higher level (e.g. 9.5 to 10.5) by
adding a small amount of base (10.0 mol/L of sodium hydroxide
solution). The supernatant turned turbid, suggesting formation of
precipitates. In order to verify charge type of the precipitates, a
water-soluble dye was added at a low concentration of 47 mg/L. The
sample color turned to strong blue of the dye color. The negative
charged dye should bind to the precipitates if the precipitates
carry positive charges because of electrostatic attraction. The
sample was centrifuged again. Blue precipitates should be observed
in bottom of the centrifuge tube if this hypothesis is correct.
Otherwise, the dye should be still dissolved in solution and the
solution remains blue dye color. FIG. 6 illustrates flocculation
using a dye indicator with and without pH adjustment. Color
difference is apparent before and after pH adjustment. Strong blue
color in FIG. 6A was observed at initial pH 7.4. Precipitates of
magnesium hydroxides did not form at this pH, where the dye was
still dissolved in solution with its color. In contrast, blue
precipitates in the bottom and colorless supernatant in FIG. 6B
were observed after pH adjustment to 10.4. The results from FIGS.
6A and 6B confirm that the positive charged precipitates were
formed at high pH 10.4. FIGS. 6A and 6B illustrates photos of dye
indicator for formation of positive charged precipitates, (A) Dye
(47 mg/L) in supernatant (pH=7.4), centrifuged again at 5,000 rpm
for 10 min. and (B) Dye (47 mg/L) in supernatant (pH=10.4),
centrifuged again at 5,000 rpm for 10 min.
Algae Harvesting through Magnetic Separation
[0069] In one exemplary method, algal cells were directly captured
by a permanent magnet in presence of particles of iron and
magnetite at different sizes and contents. In these examples,
magnetite appeared to perform better than the irons tested. Here,
three types of magnets were tested where they were all capable of
capturing algae-magnetite flocs from medium. In these experiment
configurations, a high recovery of algae (about 97%) was obtained
using a magnetite content of about 2.0% (w/v) and a strong plate
magnet. In one method, a magnetic belt conveyor was suggested for
algae harvesting in a continuous mode. An enhanced magnetic plate
using strong magnetic discs capable of use in a belt conveyor was
constructed and tested. This resulted in about 91% of the algae
captured from medium. It is possible that suspension microorganisms
can be captured from a continuous mode for a high percent recovery
of algae and high percent removal of medium. These methods could
lead to magnetic separation for suspension cultures like algae by
quick harvesting using low energy input to reduce time and cost.
These examples do not use a settling step for harvesting.
Example 2
[0070] Effects of pH on the Magnetic Flocculation
[0071] As discussed above, pH is one of the key factors in
formation of magnesium hydroxides and then further magnetic
flocculation. Effect of solution pH on the algae harvesting
efficiency is illustrated in FIG. 7. Harvesting efficiency is
defined as the difference of algae densities between raw algae
broth and the separated supernatant over the algae density of raw
broth. As expected, the solution pH had a significant influence on
the algae harvesting efficiency. When a solution pH was below 10.0,
algae harvesting was about 30%, suggesting that some of the
magnesium hydroxides did not form. Once adjusted to pH 10.0, algae
harvesting efficiency increased and reached a maximum value of 92%
at pH 11.7. Over this pH change, algae harvesting did not exhibit a
significant change. Comparing the pH profile of magnesium
hydroxides, the formation of magnesium hydroxides occurred in pH
range of 10.0 to 11.7 in FIG. 7 in this study. The results from
FIG. 7 demonstrated that magnesium hydroxide precipitates play a
key role in the algae harvesting. In addition, high harvesting
efficiency (about 92%) was obtained using this magnetic
flocculation under these conditions.
[0072] In one example, magnesium was added to a culture to a final
concentration of about 2,000 mg/L and the pH was adjusted to about
9.5. In this example, there is support that harvesting pH can be
dependent of the concentration of heavy agent provided to the
culture and this example led to a harvesting recovery of about 99%
(Table 1). Therefore, in certain exemplary methods, base addition
and pH adjustment to a more basic pH may not be needed if the
precipitating agent (e.g. magnesium) is provided at higher
concentrations. Therefore, this could eliminate or significantly
reduce costs related to supplementing the media with base for pH
adjustment.
TABLE-US-00001 TABLE 1 Magnetic flocculation at pH 9.5 at various
levels of magnesium concentrations Sample DW25-#1 DW25-#2 DW25-#3
DW25-#4 Magnesium added 498.0 994.7 1,504.9 2,032.7 (mg/L)
Fe.sub.3O.sub.4 1.0 1.0 1.0 1.0 (% w/v) Algae recovery* 27.6 38.5
91.0 99.8 (%) *Algae recovery is defined as percentage of
difference of algae densities between raw algae broth and
supernatant to algae density in raw algae broth
[0073] In certain examples, a high pH (e.g. >9) appears to be
needed for magnesium-based harvesting technology because of
formation of magnesium hydroxides. It is noted that the pH increase
can vary in expense output due in part to different bases added to
a solution. Therefore, three different bases, sodium hydroxide
(NaOH), calcium hydroxide (Ca(OH).sub.2), and ammonium hydroxide
(NH.sub.4OH), were investigated in this study.
Sodium Hydroxide Addition
[0074] Sodium hydroxide is a strong base and is soluble in water.
Sodium hydroxide solution at high concentration (e.g. 10.0 mol/L)
was added to algae broth for an increase in pH. FIG. 8 illustrates
a plot of solution pH as a function of NaOH concentrations. At pH
below 10.1, the solution pH appeared to increase linearly with
increase of NaOH concentrations, suggesting that NaOH addition was
directly proportional to the increase in solution pH. The solution
pHs between 10.3 to 11.0 only slightly increased with NaOH
addition. As discussed above, magnesium hydroxides are precipitated
at this pH range. So the NaOH added was consumed by precipitation
of magnesium hydroxides, resulting in slow increase of pH. After
this point, the solution pH increased sharply again as the
magnesium ions was consumed completely.
Ca(OH).sub.2 Addition
[0075] Unlike sodium hydroxide, calcium hydroxides (Ca(OH).sub.2)
is a relatively inexpensive source of base reagent, commonly used
to increase pH in wastewater treatment. Calcium hydroxide has low
solubility in water at pH above 10. Solid calcium hydroxide was
directly added in the algae broth in this study due to this
property. The results from the calcium hydroxide addition are
illustrated in FIG. 9. As observed, a similar trend was observed as
that of sodium hydroxides. Three algae samples were tested with
algal densities varied from 1.9-3.4 g/L. There is no significant
difference found, indicating that solution property (e.g. magnesium
concentrations), not algal densities, played a key role in the pH
changes in addition of base. FIG. 9 illustrates N. salina of 1.9
g/L; N. salina of 3.4 g/L; and N. salina of 2.1 g/L.
NH.sub.4OH Addition
[0076] Another base tested was a weak ammonium hydroxide
(NH.sub.4OH). Similar to NaOH addition, a concentrated ammonium
hydroxide solution (28-30% wt) was used for the pH adjustment. FIG.
10 illustrates that the solution pH as a function of NH.sub.4OH
concentrations. Comparing to NaOH and Ca(OH).sub.2, high NH.sub.4OH
concentration of approximate 50 mmol/L is required to reach pH 10.2
and then increased slowly. For example, solution pH was 10.3 at
NH.sub.4OH concentration of 150 mmol/L. Also, solution pH was still
10.5 when the NH.sub.4OH concentration reached at 620 mmol/L. The
results suggested that there was a low efficiency of pH increase
with NH.sub.4OH addition than that of NaOH and Ca(OH).sub.2 because
NH.sub.4OH is a weak base.
Nitrogen Stripping
[0077] Rather than base addition, inert gas (e.g. nitrogen)
stripping was also used to increase pH. Although inert gas does not
have any chemical reaction when it passes through solution,
dissolved CO.sub.2 in solution is stripped out of solution,
resulting in pH increase. In active cultures, CO.sub.2 will also be
consumed by photosynthetically active algae, again resulting in pH
increase. Nitrogen gas stripping was studied in lab tests, where
the nitrogen gas from a gas tank passed through the algae broth
(150 mL). The solution pH increased from 7.3 to 9.5 with nitrogen
stripping, indicating that inert gas stripping can be used to
increase pH. It is noted that pH of above 10 was not observed in
this experiment, even after 420 minutes exposure. This may be due
to the algae being tested in lab was not very activated for
consuming the dissolved CO.sub.2 in the media. A pH of above 10 was
obtained with air stripping when algae were grown outside under
sunlight. So inert gas (e.g. air) stripping might provide a
cost-effective way to increase pH as it does not introduce any
chemicals into media, which might not introduce any negative
influence on media recycle for algae cultivation.
Example 3
Effect of Magnetite Contents on the Magnetic Flocculation
[0078] After algal surfaces become positive charge in presence of
magnesium hydroxides, negative magnetite particles will attach to
the algae due to electrostatic attraction. Magnetite contents
affect algae harvesting performance. Effect of the magnetite
contents on the algae harvesting were shown in FIGS. 11A and 11B.
As can be seen in FIG. 11A, the magnetite content affected
significantly the harvesting efficiency. The harvesting of algae
(N. oculata of 1.7 g/L) decreased sharply when the magnetite
content was below 0.5% (w/v), suggesting that magnetite particles
were not sufficiently prevalent to attach entire algal cells in the
media. As magnetite content increased, the algae harvesting
increased slightly from 82% to 87% as the magnetite content
increased from 0.5% to 5.0% (w/v). So magnetite content should be
higher than 0.5% (w/v) in order to obtain high harvesting
performance. Similar results were obtained using N. salina of 3.4
g/L shown in FIG. 11B, confirming the influence of magnetite
contents. High efficiency of harvesting (96.9%) obtained could be
due in part to high cell density of 3.4 g/L used in this test.
Recovery of Concentrated Algae and Magnetite Particles
[0079] After the algae harvesting, the algae-magnetite particles
can be separated to obtain the concentrated algae and the magnetite
particles. One method is to decrease solution pH below the pH level
at which dissolved magnesium hydroxide precipitates. Here,
magnetite particles were separated from the media by a magnet as
electrostatic repulsion occurred between the algae and the
magnetite. FIG. 12 illustrates separation of the concentrated algae
and the magnetite at lower pH 6.6. The magnetite was captured on
the tube wall by a magnet and the concentrated algae left in the
solutions in bottom in the sample. The observation demonstrated
that the algae were concentrated by the magnetic flocculation
method through pH adjustment.
[0080] In order to investigate potential magnetite remaining in the
concentrated algae, light microscopy was used to observe the algae
before and after the magnetic recovery. FIGS. 13A-13B illustrate
microscopic images of before and after the magnetite removal in the
sample. In this Sample, pH was decreased from 10.5 (FIG. 13A) to pH
6.6 (FIG. 13B). A significant change was observed. In the higher
pH, the algae were flocculated with binding to magnetite. In the
lower pH of 6.6, the algae were separated without the flocculation
and most of the magnetite particles were removed (few magnetite
particles were found in this sample). The observations from FIG. 13
demonstrated that the magnetite particles were separated by
decreasing pH and the concentrated algae were obtained. So it is
suggested that recovered pH could be about 6.6 in order to get
concentrated algal cells essentially free of magnetite.
Regeneration and Reuse of the Magnetite Particles
[0081] It may be necessary to regenerate and reuse the magnetite
particles in order to reduce the particle cost. A set of
experiments in batch was designed to test whether the magnetite
particles can be reused. The experimental protocol is presented in
the schematic of FIG. 14. The pH of algae broth (100 mL) was
adjusted to 10.6 and dry magnetite particles (2.0 g) as 2.0% (w/v)
were added into the first broth. The sample was mixed by a vortex
mixer for one minute and the supernatant was decanted while
applying a magnet. Approximately 15 mL of water were added into the
algae-Fe.sub.3O.sub.4 paste and pH was lowered to 6.5-8.1. The
magnetite and concentrated algae were separated by holding the
magnet. The resultant magnetite particles were reused by adding
into a new batch of algae broth. In Batch #8, the resultant
magnetite before reuse was regenerated by an additional step of
water washing, lowering pH and recovering by use of the magnet.
[0082] Experimental conditions in nine batches were presented in
the Table 2. The wet magnetite particles were reused in all
experiments except the first. Harvesting pHs were at 10.6 except
that in Batch #5.
TABLE-US-00002 TABLE 2 Nine experiments of the regeneration and
reuse of magnetite Sample batch Magnetite Harvested pH Regenerated
pH #1 dry 10.6 #2* wet 10.6 8.1 #3 wet 10.6 6.8 #4 wet 10.6 7 #5
wet 10.3 6.5 #6 wet 10.6 6.6 #7 wet 10.6 6.5 #8 wet 10.6 6.5, 7.9
#9 wet 10.6 *Continuous harvesting without removal of attached
algae (N. oculata of 1.9 g/L, initial pH = 7.4)
[0083] The results of algae harvesting using reusable magnetite
were shown in FIG. 15. In general, six of nine batches (#1, #2, #3,
#4, #6, and #8) did vary slightly on harvesting efficiencies
(77-84%). Slightly lower harvesting (74%) in Batch #5 was obtained
as slight lower pH 10.3 of harvesting was used in comparison with
10.6 in other batches. However, lower harvesting efficiency of 65%
in Batch #7 and 70% in Batch #8 were obtained, suggesting that the
wet magnetite particles needed to be regenerated after the first
six batches of harvesting. The magnetite particles were washed
twice in Batch #8 and the harvesting efficiency of 83% was achieved
in Batch #9. The results from FIG. 15 demonstrate that the
magnetite particles tested can be reused without a significant loss
in harvesting efficiency after simple water washing was used for
regeneration.
Example 4
Enhancement of Sedimentation in Presence of Inorganic Particles
[0084] Alternative to the magnetic separation, gravity
sedimentation can be an inexpensive and reliable process. The
flocculated algae tied with magnetite could settle to the bottom if
the algae density is higher than that of growth media. Certainly,
the higher density of magnetite and algae will lead to higher
efficiency of sedimentation in term of shorter time and less volume
of the concentrated algae. The density of magnetite and algae
associates with physical properties of the magnetite used. Table 3
represents physical properties and the particle size distribution
of the magnetite particles (e.g. Pirox 200) used in this study. The
specific gravity of the particles is 5.23 g/cm.sup.3, which tends
to gravity sedimentation. The particle sizes are smaller than 4.0
microns in 90% of the particles and smaller than 2.0 microns in 50%
of the particles. Particles of very small sizes do not tend to
sedimentation although they provide more specific surface area (3.0
m.sup.2/g) for the algae attachment.
TABLE-US-00003 TABLE 3 Physical properties of magnetite powder
(Fe.sub.3O.sub.4) from Pirox 200 Bulk Density 737 kg/m.sup.3
Specific Gravity 5.23 g/cm.sup.3 Specific Surface Area 3.0
m.sup.2/g Numeric Particle Size D50 2.0 .mu.m D90 4.0 .mu.m
[0085] In addition, a ratio of magnetite to algae (or content of
magnetite for a specific density of algae broth) will also affect
the algal sedimentation. FIG. 16 represents effects of magnetite
contents on the algae sedimentation by gravity. The water removed
is defined as the separated supernatant volume over the total
volume of raw algal broth. The water removed was significantly
increased with increasing content of magnetite from 0 to 5.0%
(w/v). The settling performance has significantly changed when the
magnetite contents were below 2% (w/v). After a range of 2% to 5%,
there was only a slight difference, for time (e.g. >140 minute).
Settling performance was significantly improved with time. The
highest amount of water removed, about 74%, was obtained when the
magnetite contact was higher than 2.0% (w/v) at 208 minutes. FIG.
16 represents sedimentation by gravity in presence of the magnetite
(N. oculata of 1.7 g/L, settling pH 10.6).
[0086] In order to investigate particle density on influence of the
settling performance, another high density material of non-magnetic
metal particles was selected, tungsten (W). Tungsten in this test
has a density of 19.3 g/cm.sup.3 with a particle size of about
0.6-1 .mu.m. The sedimentation performance varied as shown in FIG.
17. Similar results compared to the magnetite sedimentation profile
were obtained with the use of tungsten. The highest water removal
was 84% in this study when the tungsten content was 5.0% (w/v) at
206 minutes of the settling time, which was greater than that of
74% using the magnetite demonstrated in FIG. 16. Sedimentation by
gravity in presence of the tungsten (N. oculata of 1.7 g/L,
settling pH 10.6) is also represented here.
[0087] To compare magnetite and tungsten regarding sedimentation,
two particle contents (0.5% and 2.0%) were selected. The results
are presented in FIG. 18. Higher extent water removal using
tungsten was shown than that using magnetite in both concentrations
of particles, demonstrating that tungsten has superior performance
of sedimentation compared to magnetite. The results confirmed that
particle density plays a role in the sedimentation when even lower
particle size of tungsten (0.6-1 .mu.m) was used in comparison to
the magnetite in bigger sizes (see Table 2). FIG. 18 represents a
comparison of magnetite and tungsten on sedimentation by gravity
(N. oculata of 1.7 g/L, settling pH 10.6).
Effect of Particle Size on Algae Settling by Gravity
Enhancement of Sedimentation in the Presence of Inorganic
Particles
[0088] In other exemplary methods, different particle sizes were
tested for their effects on sedimentation of suspension cultures
described herein. For example, particles of iron and silica in
three different sizes ranging from several micrometers to hundred
micrometers were selected as model systems in an algal model. Both
iron and silica can enhance the algae settling within certain
limitations of particle sizes. In these examples, very low dry mass
of about 0.04 g/L in supernatant was obtained in comparison to raw
dry mass of 3.7 g/L. Iron particles in three different sizes were
tested in order to determine size limitation. For algae settling in
presence of iron particles, particle sizes in 6-9 .mu.m and about
44 .mu.m demonstrated clear settling at about 60 min. The dry
masses in the supernatants were 0.02 g/L and 0.04 g/L,
respectively, indicating that about 99% of algae were settled in
the slurry in comparison to the dry mass of 3.7 g/L in raw algae
broth. It was observed that no significant difference in settling
was documented for these test sizes even though one used about 44
.mu.m particles, much bigger than the other at 6-9 .mu.m. Iron
particles in size of 10-40 mesh (420-2,000 .mu.m) did not appear to
significantly enhance the algae settling in these examples. Silica
in size of 50-70 mesh (297-210 .mu.m) also did not seem to enhance
settling. These larger particles did not attach to algae when they
mixed with algae, likely because they settled alone by gravity
without associating with algae. Of these particles tested, iron
material may be one good candidate for algae harvesting because of
its magnetic properties and inexpensive material cost. It is
contemplated that the particle size can be about 1 .mu.m to about
100 .mu.m, in order to enhance settling of a suspension
culture.
TABLE-US-00004 TABLE 4 Densities and sizes of particles tested
Particle Density (g/ml) Size Iron 7.86 (1) 6-9 .mu.m (2) ~325 mesh
(~44 .mu.m) (3) 10-40 mesh (2,000-420 .mu.m) Silica 2.6 (1) 0.5-10
.mu.m (80% between 1-5 .mu.m) (2) <230 mesh (<63 .mu.m) (3)
50-70 mesh (297-210 .mu.m) Tungsten 19.3 (1) 0.6-1 .mu.m Magnetite
4.8-5.2 (1) <5 .mu.m (2) <10 .mu.m (50% less than 2 .mu.m,
90% less than 4 .mu.m)
Example 5
Process Design and Continuous Harvesting Algae
Process Design
[0089] In one exemplary experiment, a new process was developed
based on the magnetic flocculation. A schematic of flow chart of
the harvesting process is shown in FIG. 19. Algae broth will be
sent to Tank #1 for increasing pH and adding iron oxides. Mixing in
retention time of 5-10 minutes is required to make uniform
dispersion of iron oxides in the Tank #1 and the mixed algae will
pass through a Magnetic Separator. The magnetite and attached algae
will be settled by gravity in a Settler #1 in retention time of
15-60 minutes, aiming at 75-90% (v/v) of supernatant returning to
the algae cultivation system. The remaining 10-25% (v/v)
flocculated algae-iron oxides will be transferred to Tank #2 for pH
decrease. The resulting algae will be sent to Settler #2. In
another example, the recovered iron oxides might need an additional
step of water washing for regeneration and then will be sent back
to Tank #1 for reuse. The resultant algae concentrates will be sent
to further processing for lipid extraction.
[0090] It is noted that the general magnetic separator used for
separation is not limited to the particular type of Magnetic
Separator used here. It could be equipment which provides a
magnetic field for enhancement of algae sedimentation. Also, the
Magnetic Separator may be placed next to the Settler #1. In
addition, the Magnetic Separator could be removed.
Example 6
Continuous Harvesting Algae
Role of Magnetite Particles in the Magnetic Harvesting
[0091] In order to examine influence of magnetite particles,
another experiment using a magnetic separator was designed as
illustrated in FIG. 20. Algae broth (N. salina of 3.44 g/L, 1.0-1.3
liter) was mixed by mechanical stirring at about 1,000 rpm for
about 5 min in presence of magnetite (2.0% w/v) or in absence of
magnetite. The algae broths were passed through a Magnetic
Separator with permanent magnets (Model PQ-2, S. G. Frantz Co.
Inc.) at a flow rate of 100 ml/L. The resultant algae were settled
by gravity. Three tests were conducted under experimental
conditions presented in Table 5. Four samples were taken for dry
mass determination for evaluation of harvesting efficiency. Each
example was collected in Test #1 and Test #2 after the tests were
completed. Two samples were taken at 2.5 and 4.0 minutes after the
algae were passed through the Magnetic Separator.
TABLE-US-00005 TABLE 5 Three experiments with the Magnetic
Separator in presence or absence of magnetite Magnetite Sampled
time Dry mass Algae Test pH (% w/v) (min) (g/L).sup.b harvesting
(%) #1 7.3 0 finished.sup.a 3.12 9.3 #2 10.6 0 finished.sup.a 2.81
18.3 #3 10.6 2.5 2.5 0.10 97.1 4.0 0.08 97.8 .sup.aThe samples were
taken after the experiments were completed. .sup.bAll samples were
settled about 30 minutes and then dry mass in the supernatants were
determined.
[0092] In both Test #1 and Test #2, a significant color change was
not observed, after the (bright green) algae were passed through
the Magnetic Separator. This observation suggested that the
Magnetic Separator did not capture a lot of algae even when the
algae were flocculated by magnesium hydroxides at pH 10.6 in Test
#2. However, significant color changes were observed in Test #3.
Almost colorless effluent was first found at about 2 minutes,
indicating that the algae were captured by the Magnetic Separator.
This is confirmed by the fact that the captured algae were found
inside the Magnetic Separator when the Separator was opened. After
running about 2.5 minutes, the algae flowed out the system,
indicating that the Separator had reached a capture capacity in
this test.
[0093] The harvesting results were presented in the Table 5. The
day mass of 3.12 g/L in Test #1 was slightly lower than that of raw
algae of 3.44 g/L, suggesting that a small amount of algae was
adsorbed in the Separator. In addition of the adsorption, the
flocculated algae in Test #2 had further slight sedimentation and
resulted in lower dry mass of 2.81 g/L. However, sharp reductions
in the dry mass (0.10 and 0.08 g/L) were obtained in both samples
of Test #3. High algae harvesting efficiency (>97%) was found in
Test #3. From the results of three tests, it is concluded that
magnetite particles indeed enhanced the algae harvesting using the
Magnetic Separator.
Example 7
[0094] Enhancement of Algae Sedimentation by Magnetic Separator
[0095] Sedimentation of the algae-magnetite flocs is enhanced by a
magnetic field. When the broth containing algae-magnetite passes
through a magnetic field, the algae-magnetite flocs become
magnetized and attached to each other and formed larger flocs. The
larger flocs have enough mass to cause them to settle out of the
media at a much faster rate than initial algae-magnetite flocs.
Experiments were conducted to verify this hypothesis using same
procedure in FIG. 20. The only difference was to withdraw one more
sample (Separator Influent) without passing the Magnetic Separator
in order to compare with the sample (Separator Effluent) which had
passed through the Magnetic Separator.
[0096] However, the colorful algae flowed out the Magnetic
Separator after about 9 minutes. This observation suggested that
the Magnetic Separator had reached saturation of the algae capture
capacity. Longer time of 8.5 minutes for clear effluent in this
test was observed than that (about 2 minutes) of the test (Example
6) discussed previously. There may be lower algae density (e.g.
2.06 g/L) in this test in comparison to the previous test (e.g.
3.44 g/L). The ratio of magnetite to algae density may influence
the capture capacity of the Magnetic Separator.
[0097] However, the colorful algae flowed out the Magnetic
Separator after about 9 minutes. This observation suggested that
the Magnetic Separator has reached saturation of the algae capture.
Longer time of 8.5 minutes for clear effluent in this test was
observed than that (about 2 minutes) of the test discussed
previously. There may be lower algae density (e.g. 2.06 g/L) in
this test in comparison to the previous test (e.g. 3.44 g/L). The
ratio of magnetite to algae density may influence the capture cap
of the Magnetic Separator.
[0098] 100 ml of effluent after 9 minutes was collected to start
sedimentation by gravity in comparison to another sample, influent,
without passing through the magnetic separator. FIG. 20 illustrates
photos of two samples in various settling time. Significant
differences were observed between the two samples. The effluent
settled much faster than the influent in the time period tested.
This observation demonstrated that the magnetic field provided by
the Magnetic Separator significantly enhanced the algae
sedimentation due to magnetization of the magnetic algae. For
example, the effluent separated about 85 ml of supernatant in
comparison with about 10 ml of supernatant separated in the
influent in 10.5 minutes. About 90 ml of supernatant was separated
in the effluent in settling 31 minutes, indicating that about 90%
of water can be removed by this system. FIG. 20 represents
enhancement of algae settling by a magnetic field. Left sample:
influent (marked "No Treatment" (NT)); Right sample: effluent
(marked "Treated" (T)).
[0099] In order to observe the changes of algal cells during the
harvesting process, the microscopic images were taken under a light
microscopy. FIGS. 21A-21D illustrate the microscopic images of the
algae during the harvesting process. The flocculated algae were
observed in FIGS. 21A-21D due to high pH 10.6. The magnetite
particles were attached with the flocculated algae in the influent
before the magnetic separator in FIGS. 21A-21D. Similar images were
seen in the effluent before settling in FIG. 21C. FIG. 21D
illustrates higher density of algae-magnetite flocs in the effluent
after settling. The algae were recovered by lowering the pH to 6.7.
All the images confirmed the concept of the magnetic harvesting
method proposed above.
[0100] Magnetic dosage factor and concentration factor variable can
be considered for flocculation/coagulation. In certain methods, a
1% magnetite dosage factor can be of use to coagulate suspension
cultures of compositions disclosed herein. In other methods,
settling time of a culture exposed to magnetite or other agent of
use to settle suspension cultures can be considered.
Example 8
[0101] A bench-scale unit was designed and set up to test
continuous harvesting of algae. A schematic flow chart of algae
harvesting is represented in FIG. 22. One test was conducted using
this unit.
[0102] Algae (N. salina) broth (104.8 liters) were pumped into the
Mix Tank #1 (5 liter volume) at a flow rate of 1.0 liter/min with a
retention time of 5 minutes. Sodium hydroxide (NaOH) solution (5.0
mol/L) and magnetite (Fe.sub.3O.sub.4) slurry (35.5% w/w) were
flowed into the Mix Tank #1, respectively. The solution pH was kept
10.50-10.61 and magnetite content was 1.05% (w/v) of algae feed.
Mechanical mixing was conducted in the Mix Tank #1 at approximately
900 rpm. The algae-magnetite mixture was sent to the Settling Tank
#1 at an overflow retention of about 60 minutes. The clear
supernatant was overflowed to a Magnetic Separator to remove
remaining magnetite and then to an Effluent Tank. The settled
algae-magnetite mixture was under flowed into the Mix Tank #2
(volume of 5 liters). The solution pH was adjusted to between
6.47-7.19 using an addition of hydrochloric acid (HCl) at 3.0 mol/L
solution into Mix Tank #2. Mechanical mixing was conducted in the
Mix Tank #2 at approximate 900 rpm. The resultant mixed
algae-magnetite mixture was pumped into the Settling Tank #2 for
separation of concentrated algae and magnetite. The thickened algae
were overflowed and magnetite slurry was under flowed from the
Settling Tank #2. Thus, the concentrated algae and magnetite were
recovered.
[0103] In this exemplary method, flow cytometry was used to assess
algal cultures having been harvest by iron flocculation compared to
cultures prior to iron flocculation (see FIG. 23). Table 6 and
Table 7 illustrate the results of mass balances of algae and water.
The results demonstrated that the majority of water (about 75%) was
removed and so algae were concentrated by a factor of 4.0. The
algae density in supernatant effluent was very low at 0.0004 g/L,
indicated that algal loss in the supernatant at 0.017%. Thus algae
recovery was over 99% obtained in this test. A schematic flow chart
of algae harvesting is represented in FIG. 22.
TABLE-US-00006 TABLE 6 Algae mass balance Volume Dry mass Dry
biomass (liter) (g/L) (g) Raw Algae #1 90.8 1.292 117.3 Raw Algae
#2 50.0 2.888 144.4 Recovery Algae #1 18.93 5.840 110.6 Recovery
Algae #2 16.28 5.160 84.0 Algae #3 in recovered magnetite 3.4 17.14
58.3 slurry Supernatant 113.17 0.0004 0.045 Total amount in
effluent (g) 252.9 Total amount in influent (g) 261.7 Algae loss in
supernatant (%) 0.017 Error (%) -3.4
TABLE-US-00007 TABLE 7 Water volume balance Volume (liter) Influent
Water from raw algae broth 140.8 Water from magnetite slurry 2.68
Water from base solution 0.79 Water from acid solution 1.38
Influent Total 145.7 Effluent Water from supernatant 113.17 Water
in recovered algae 35.2 Water in recovered magnetite slurry 2.96
Effluent Total 151.3 Error (%) +3.8
Example 9
[0104] Removal of Magnetite Residues from Settled Supernatant
[0105] In another exemplary experiment, methods for recycling and
reusing media are contemplated. Magnetite residues in supernatant
were observed after gravity settling under a microscope. Medium pH
and associated magnesium hydroxides on the magnetite settling were
investigated. The results suggest that magnesium hydroxide
precipitates might be a negative factor on the magnetite settling
in terms of water removal. It was attempted to remove magnetite
residues from the supernatant using pH adjustment, reuse of
magnetite and magnetic capture. Magnetite residues were not
completely removed at low pH by reusing magnetite particles.
Further removal of the magnetite residues was attempted by magnetic
capture through a strong magnetic field formed by two permanent
magnets. Supernatant containing very few magnetite particles or
essentially magnetite-free could be obtained after the magnetic
capture. It is suggested to use a high gradient magnetic filter in
scale-up operation. Medium could be recycled for algae cultivation
after the magnetite residues are removed.
Use of Stronger Magnetic Field
[0106] In one example, a strong magnetic field was used to capture
magnetite from a supernatant. In this example, a strong magnetic
field could be formed using two magnets (e.g. permanent magnet from
Bangs Laboratories Inc.) with a flat or compressed vessel secured
between the magnets as demonstrated in FIG. 24. Using this type of
set-up, supernatant sample was added to the vessel and left for
about 15 min (e.g. aged). The essentially clear supernatant was
decanted while maintaining the set-up while the magnetite residues
were captured within the walls of the vessel for recovery of
magnetite. These methods could be used for rapid consolidation and
harvesting of a culture for testing or scale-up for harvesting and
reuse of media etc. In addition, the treated supernatants were
centrifuged (for example at 6,000 rpm for 10 min) to get
concentrated samples for microscopic observation and potential
testing.
[0107] Some magnetite particles were captured by the strong
magnetic field as demonstrated in FIG. 24, indicating that the
magnets under the strong magnetic field can capture magnetite
residues from the supernatant. FIGS. 25A and 25B illustrate an
attempt to observe potential particle residues under a microscope
using two representative samples (25A and 25B). No magnetite
particles were observed in the images, suggesting that very few
magnetite particles or free magnetite were present in the treated
supernatant samples because they were essentially removed by
harvesting.
Materials and Methods
[0108] In certain methods, various chemicals, procedures and
materials may be used, including, but not limited to, the
following:
Examples of Algae: Nannochloropsis oculata, Nannochloropsis salina
Magnetite [Iron (II, III) oxide] powder (<5 .mu.m) from Aldrich
(e.g. Cat #: 310069) Magnetite [Iron (II, III) oxide] powder from
Pirox 200 Tungsten (W) (Sigma-Aldrich; e.g. Cat #: 510106) Reactive
Blue 4 (Sigma-Aldrich; e.g. Cat #: 244813) Calcium hydroxide
(Ca(OH).sub.2) (Fluka; e.g. Cat #: 21181) Ammonium hydroxide
(NH.sub.4OH) (Sigma-Aldrich; e.g. Cat #: 221228) Permanent magnet
from Bangs Laboratories Inc. (e.g. BioMag Flask Separator, Cat #:
MS004) Magnetic Separator of Permanent Magnet from S. G. Frantz Co.
Inc. (Model PQ-2) Algal growth:
For Example 8:
[0109] Raw algae (e.g. N. salina) reserved in tanks with air
bubbling under lamps in Example 8 Magnetite stock slurry: 35.5%
(w/w)--magnetite addition of 1.05% (w/v) of algae feed Supernatant
passed through a Magnetic Separator pH: harvesting pH=10.50-10.61;
recovered pH=6.47-7.19 Influent flow rate: 1.0 L/min
[0110] All of the COMPOSITIONS and/or METHODS and/or APPARATUS
disclosed and claimed herein can be made and executed without undue
experimentation in light of the present disclosure. While the
compositions and methods of this invention have been described in
terms of preferred embodiments, it will be apparent to those of
skill in the art that variation may be applied to the COMPOSITIONS
and/or METHODS and/or APPARATUS and in the steps or in the sequence
of steps of the method described herein without departing from the
concept, spirit and scope of the invention. More specifically, it
will be apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
* * * * *