U.S. patent application number 16/044419 was filed with the patent office on 2019-02-07 for microalgal compositions and uses thereof.
The applicant listed for this patent is Corbion Biotech, Inc.. Invention is credited to Adrienne McKee, John Piechocki, Celine Schiff-Deb, Garrett Sell, Staci Springer, Bryce A.R. Sullivan.
Application Number | 20190040334 16/044419 |
Document ID | / |
Family ID | 55702109 |
Filed Date | 2019-02-07 |
United States Patent
Application |
20190040334 |
Kind Code |
A1 |
Schiff-Deb; Celine ; et
al. |
February 7, 2019 |
MICROALGAL COMPOSITIONS AND USES THEREOF
Abstract
Provided are microalgal compositions and methods for their use.
The microalgal compositions include lubricants that find use in
industrial and other applications.
Inventors: |
Schiff-Deb; Celine; (South
San Francisco, CA) ; McKee; Adrienne; (South San
Francisco, CA) ; Piechocki; John; (South San
Francisco, CA) ; Springer; Staci; (South San
Francisco, CA) ; Sell; Garrett; (South San Francisco,
CA) ; Sullivan; Bryce A.R.; (South San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Corbion Biotech, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
55702109 |
Appl. No.: |
16/044419 |
Filed: |
July 24, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15080458 |
Mar 24, 2016 |
10053646 |
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16044419 |
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62175014 |
Jun 12, 2015 |
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62162553 |
May 15, 2015 |
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62137784 |
Mar 24, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C10M 2207/2805 20130101;
C10N 2010/08 20130101; C10M 2207/401 20130101; C10M 159/08
20130101; C10N 2030/62 20200501; C10N 2030/64 20200501; C10N
2050/08 20130101; C10M 2207/046 20130101; C10M 159/02 20130101;
C10N 2040/08 20130101; C10M 173/00 20130101; C10N 2040/12 20130101;
C10N 2040/38 20200501; C10M 2209/1033 20130101; C10M 129/40
20130101; C10M 2209/1045 20130101; C10M 2207/301 20130101; C10N
2020/06 20130101; C10N 2040/20 20130101; C10M 2201/041 20130101;
C10N 2040/02 20130101; C10M 2201/065 20130101; C10N 2050/10
20130101; C10M 2201/066 20130101; C10M 169/04 20130101; C10N
2020/013 20200501; C10N 2040/30 20130101; C10M 129/20 20130101;
C10N 2040/24 20130101; C10N 2030/06 20130101; C10N 2020/081
20200501; C10M 2203/1065 20130101; C10M 2207/404 20130101; C10N
2040/22 20130101; C10M 2207/40 20130101; C10N 2020/055 20200501;
C10N 2040/04 20130101; C10N 2040/14 20130101; C10N 2040/36
20130101; C10M 2201/061 20130101; C10M 2203/1006 20130101; C10N
2010/12 20130101; C10N 2040/00 20130101; C10N 2040/25 20130101;
C10M 2209/1055 20130101 |
International
Class: |
C10M 159/02 20060101
C10M159/02; C10M 129/40 20060101 C10M129/40 |
Claims
1.-31. (canceled)
32. A method for providing lubrication to a surface, the method
comprising applying a lubricant to the surface, the lubricant
comprising oleaginous microbial biomass, wherein the oleaginous
microbial biomass comprises at least 20% lipid by dry cell weight,
said lubricant further comprising at least one other ingredient
selected from the group consisting of anti-oxidant, corrosion
inhibitor, metal deactivator, binder, chelating agent, oxygen
scavenger, anti-wear agent, extreme pressure resistance additive,
antimicrobial agent, pH adjuster, emulsifier, lubricity agent,
vegetable oil, petroleum derived oil, high viscosity petroleum
hydrocarbon oil, petroleum derivative, pour point depressant,
moisture scavenger, defoamers, anti-misting agent, odorant,
surfactant, humectant, rheology modifier, and colorant.
33. The method of claim 32, wherein the oleaginous biomass is
unlysed cells and wherein the unlysed cells comprise at least 35%
lipid by dry weight.
34. The method of claim 32, wherein the oleaginous biomass
comprises at least 50% lipid.
35. The method of claim 32, wherein the antimicrobial agent is
selected from the group consisting of 1,2-Benzisothiazolin-3-one,
sodium omadine, phenolics, p-chloro-m-cresol, halogen substituted
carbamates, isothiazolone derivatives, bromonitriles
dinitromorpholines, amphotericin, triazine, BIT, MIT, potassium
sorbate, sodium benzoate, and glutaraldehyde.
36. The method of claim 35, wherein the antimicrobial agent is
present in the lubricant at a concentration of 0.0005% to 6% by
weight.
37. The method of claim 32, wherein the rheology modifier is
selected from the group consisting of hydroxyethyl cellulose,
carboxymethyl cellulose, xanthan gum, guar gum, starch, and
polyanionic cellulose.
38. The method of claim 37, wherein the rheology modifier is
present in the lubricant at a concentration of 0.005% to 5% by
weight.
39. The method of claim 32, wherein the lipid provided by the
oleaginous microbial biomass comprises 15% C16:0 and greater than
55% 18:1.
40. The method of claim 32, wherein the oleaginous microbial
biomass is microalgae.
41. The method of claim 32, wherein the microalgae is of the genus
Prototheca, Auxenochlorella, Chlorella, or Parachlorella.
42. The method of claim 41, wherein the microalgae is of the genus
Prototheca.
43. The method of claim 42, wherein the microalgae is Prototheca
moriformis.
44. A method for providing lubrication to a surface, the method
comprising applying a lubricant to the surface, the lubricant
comprising oleaginous microalgal biomass, wherein the oleaginous
microalgal biomass is unlysed cells and the unlysed cells comprise
at least 20% lipid by dry cell weight, said lubricant further
comprising at least one other ingredient selected from the group
consisting of anti-oxidant, corrosion inhibitor, metal deactivator,
binder, chelating agent, oxygen scavenger, anti-wear agent, extreme
pressure resistance additive, antimicrobial agent, pH adjuster,
emulsifier, lubricity agent, vegetable oil, petroleum derived oil,
high viscosity petroleum hydrocarbon oil, petroleum derivative,
pour point depressant, moisture scavenger, defoamers, anti-misting
agent, odorant, surfactant, humectant, rheology modifier, and
colorant.
45. The method of claim 44, wherein the antimicrobial agent
selected from the group consisting of 1,2-Benzisothiazolin-3-one,
sodium omadine, phenolics, p-chloro-m-cresol, halogen substituted
carbamates, isothiazolone derivatives, bromonitriles
dinitromorpholines, amphotericin, triazine, BIT, MIT, potassium
sorbate, sodium benzoate, and glutaraldehyde.
46. The method of claim 44, wherein the rheology modifier is
selected from the group consisting of hydroxyethyl cellulose,
carboxymethyl cellulose, xanthan gum, guar gum, starch, and
polyanionic cellulose.
47. The method of claim 44, wherein the microalgae is of the genus
Prototheca, Auxenochlorella, Chlorella, or Parachlorella.
48. The method of claim 44, wherein the microalgae is of the genus
Prototheca.
49. The method of claim 44, wherein the microalgae is Prototheca
moriformis.
50. A method for providing lubrication to a surface, the method
comprising applying a lubricant to the surface, the lubricant
comprising oleaginous microalgal biomass, wherein the oleaginous
microalgal biomass is unlysed cells of the genus Prototheca,
wherein the oleaginous microalgal biomass comprises at least 20%
lipid by dry cell weight, said lubricant further comprising at
least one other ingredient selected from the group consisting of
anti-oxidant, corrosion inhibitor, metal deactivator, binder,
chelating agent, oxygen scavenger, anti-wear agent, extreme
pressure resistance additive, antimicrobial agent, pH adjuster,
emulsifier, lubricity agent, vegetable oil, petroleum derived oil,
high viscosity petroleum hydrocarbon oil, petroleum derivative,
pour point depressant, moisture scavenger, defoamers, anti-misting
agent, odorant, surfactant, humectant, rheology modifier, and
colorant.
51. The method of claim 1, wherein the oleaginous microalgal
biomass is Prorotheca moriformis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/080,458, titled "MICROALGAL COMPOSITIONS
AND USES THEREOF", filed Mar. 24, 2018, which claims the benefit
under 35 USC 119(e) of U.S. Provisional Patent Application No.
62/137,784, filed Mar. 24, 2015, U.S. Provisional Patent
Application No. 62/162,553, filed May 15, 2015, and U.S.
Provisional Patent Application No. 62/175,014, filed Jun. 12, 2015,
each of which is incorporated herein by reference in its
entirety.
BACKGROUND
[0002] Solid or dry film lubricants function as friction reducers
between moving surfaces. Common solid lubricants include molybdenum
and tungsten disulfide, boron nitride, and graphite. A need exists
for alternative and improved solid lubricants.
SUMMARY
[0003] The present disclosure provides microalgal compositions and
methods for their use.
[0004] In one embodiment, provided is a lubricant comprising an
oleaginous microbial biomass, wherein the oleaginous microbial
biomass comprises intact cells containing at least 50% triglyceride
oil.
[0005] In another embodiment, provided is a floor sweep composition
comprising an oleaginous microbial biomass, wherein the oleaginous
microbial biomass comprises intact cells containing at least 50%
triglyceride oil.
[0006] In one embodiment, provided is a material suitable for use
in 3D printing comprising an oleaginous microbial biomass. In some
embodiments, provided is an object printed using a 3D printing
material comprising an oleaginous microbial biomass. In some
embodiments, the 3D printing material is in a powder form. Such a
form can be readily used when a sintering process is being used to
print an object. The material can also be in a filament form, such
as that suitable for printing using fused deposition modeling
(FDM). In some embodiments, the microalgal biomass comprises 1 to
85% by weight of the 3D printing material. In other embodiments,
the microalgal biomass comprises at least 5%, 10%, 15%, 20%, or 25%
by weight of the 3D printing material. In some embodiments, the 3D
printing material comprises microalgal biomass and a thermoplastic.
In some embodiments, the thermoplastic is Polylactic Acid (PLA) or
Acrylonitrile Butadiene Styrene (ABS). In some embodiments of the
3D printing material, the microalgal biomass comprises intact
cells.
[0007] In some embodiments, the lubricant is selected from the
group consisting of a spray oil, food grade lubricant, a railroad
lubricant, a gear lubricant, a bearing lubricant, crankcase
lubricant, a cylinder lubricant, a compressor lubricant, a turbine
lubricant, a chain lubricant, an oven chain lubricant, wire rope
lubricant, a conveyor lubricant, a combustion engine lubricant, an
electric motor lubricant, a total-loss lubricant, a textile
lubricant, a heat transfer fluid, a release agent, a hydraulic
fluid, a metal working fluid, and a grease.
[0008] In some embodiments, the lubricant comprises one or more of
an anti-oxidant, a corrosion inhibitor, a metal deactivator, a
binder, a chelating agent, a metal chelator, an oxygen scavenger,
an anti-wear agent, an extreme pressure resistance additive, an
anti-microbial agent, a biocide, a bacteriocide, a fungicide, a pH
adjuster, an emulsifier, a lubricity agent, a vegetable oil, a
petroleum derived oil, a high viscosity petroleum hydrocarbon oil,
a petroleum derivative, a pour point depressant, a moisture
scavenger, a defoamers, an anti-misting agent, an odorant, a
surfactant, a humectant, a rheology modifier, or a colorant.
[0009] In some embodiments, the lubricant is a metal working fluid.
In other embodiments, the metal working fluid is a cutting
lubricant, a gun drilling lubricant, stamping lubricant, a metal
forming lubricant, and a way lubricant. In still other embodiments,
the lubricant comprises one or more of a napthenic oil, a paraffinc
oil, a fatty acid ester, a high molecular weight ester, a glycol
ester, an ethylene oxide copolymer, a polypropylene oxide
copolymer, a naturally occurring triglyceride, graphite, graphite
fluoride, molybdenum disulfide, tungsten disulfide, tin sulfide,
boron nitride.
[0010] In some embodiments, the oleaginous biomass comprises at
least 90%, 80%, 70%, 60%, or 50% intact cells.
[0011] In some embodiments, the intact cells comprise at least 60%,
65%, 70%, 80%, 85%, or 90% triglyceride oil.
[0012] In some embodiments, the lubricant or compositions provided
herein further comprises lysed cells.
[0013] In some embodiments, the oleaginous microbial biomass is
obtained from a microalgae.
[0014] In some embodiments, the microalgae is of the genus
Prototheca, Auxenochlorella, Chlorella, or Parachlorella. In other
embodiments, the microalgae is of the species Prototheca
moriformis. In still other embodiments, the microalgae is of the
species Auxeochlorella protothecoides.
[0015] In some embodiments, the triglyceride oil has fatty acid
profile has at least 75%, 80%, or 85% C18:1.
[0016] In some embodiments, the oil has a fatty acid profile of
greater than 85% C18:1 and less than 3% polyunsaturates.
[0017] In some embodiments, the oil has a fatty acid profile has
less than 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,
0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% polyunsaturated fatty
acids.
[0018] In some embodiments, the oil has a fatty acid profile of
greater than 15% C16:0 and greater than 55% 18:1.
[0019] In some embodiments, the oil has a fatty acid profile of
greater than 50%, 60%, 70%, or 80% combined C10:0 and C12:0.
[0020] In some embodiments, the oil has a fatty acid profile of
greater than 60% C10:0 and C12:0 and greater than 10% C14:0.
[0021] In some embodiments, the oil has a fatty acid profile of
greater than 40%, 45%, or 50% C14:0.
[0022] In some embodiments, the oil has a fatty acid profile of at
least 70% SOS and no more than 4% trisaturates.
[0023] In some embodiments, the oil has a fatty acid profile of
greater than 50% C18:0 and greater than 30% C18:1.
[0024] In some embodiments, provided is a method for providing
lubrication to a surface, the method comprising applying a
lubricant disclosed herein to the surface.
[0025] In some embodiments, the surface is a metal. In other
embodiments, the lubricant reduces metal on metal friction.
[0026] In some embodiments, the lubricant forms a film on the
surface.
[0027] In some embodiments, the lubricant is an oil based
lubricant. In some embodiments, the lubricant is water based
lubricant. In some embodiments, the oil based lubricant contains
5-25% water.
[0028] In some embodiments, the lubricant comprises predominantly
intact cells. In some embodiments, more than 50% of the cells are
intact. In some embodiments, more than 75% of the cells are intact.
In some embodiments, more than 90% of the cells are intact.
[0029] In some embodiments, the lubricant comprises predominantly
lysed cells. In some embodiments, at least 75% of the cells by
weight are lysed. In some embodiments, at least 85% of the cells by
weight are lysed. In some embodiments, at least 90% of the cells by
weight are lysed.
[0030] In some embodiments, the lubricant comprises delipidated
cells. In some embodiments, at least 70% by weight of oil has been
extracted. In some embodiments, at least 80% by weight of oil has
been extracted. In some embodiments, at least 85% by weight of oil
has been extracted. In some embodiments, at least 90% by weight of
oil has been extracted from the cells.
[0031] In some embodiments, the delipidated cells are treated with
acid and/or base. The acid and/or base treatment digests the
cells.
[0032] In the various lubricants and/or methods discussed above and
herein, solid particles in the lubricant can contribute to the
lubricant's lubricity. In some cases, the solid particles have a
particle size distribution d50 value of from 100 to 500 .mu.m,
wherein the d50 value is the median diameter of particle size
distribution at 50% of the distribution, where 50% of the particles
are above the d50 value and 50% are below the d50 value. For
example, for a sample with a particle size distribution of d50 of
100 .mu.m, 50% of the particles are greater than 100 .mu.m and 50%
of the particles are less than 100 .mu.m. In some embodiments, the
d50 value is from 200 to 400 .mu.m. In some embodiments, the d50
value is from 300 to 400 .mu.m. For a sample with a particle size
distribution of d10 of 100 .mu.m, 90% of the particles are greater
than 100 .mu.m and 10% of the particles are less than 100 .mu.m.
Similarly, for a sample with a particle size distribution of d90 of
100 .mu.m, 10% of the particles are greater than 100 .mu.m and 90%
of the particles are less than 100 .mu.m.
[0033] In some embodiments, provided is a water based lubricant
comprising predominantly intact cells. In some such embodiments,
the lubricant has a particle size distribution d50 value of from 5
to 30 .mu.m. In some such embodiments, the lubricant has a particle
size distribution d50 value of from 7 to 12 .mu.m.
[0034] In some embodiments, provided is an oil based lubricant
comprising predominantly intact cells. In some such embodiments,
the lubricant has a particle size distribution d50 value of from
100 to 500 .mu.m. In some such embodiments, the lubricant has a
particle size distribution d50 value of from 100 to 250 .mu.m.
[0035] In some embodiments, provided is a water based lubricant
comprising predominantly lysed cells. In some such embodiments, the
lubricant has a particle size distribution d50 value of from 0.5 to
15 .mu.m. In some such embodiments, the lubricant has a particle
size distribution d50 value of from 6 to 12 .mu.m.
[0036] In some embodiments, provided is an oil based lubricant
comprising predominantly lysed cells. In some such embodiments, the
lubricant has a particle size distribution d50 value of from 5 to
20 .mu.m. In some such embodiments, the lubricant has a particle
size distribution d50 value of from 8 to 14 .mu.m.
[0037] In some embodiments, provided is a water based lubricant
comprising delipidated cells. In some such embodiments, the
lubricant has a particle size distribution d50 value of from 0.5 to
20 .mu.m. In some such embodiments, the lubricant has a particle
size distribution d50 value of from 5 to 15 .mu.m.
[0038] In some embodiments, provided is an oil based lubricant
comprising delipidated cells. In some such embodiments, the
lubricant has a particle size distribution d50 value of from 0.5 to
200 .mu.m. In some such embodiments, the lubricant has a particle
size distribution d50 value of from 10 to 100 .mu.m.
[0039] In the various lubricants and/or methods discussed above and
herein, the lubricant can have a decreased health risk (e.g. health
risk due to inhalation) compared to traditional solid film
lubricants such as those containing graphite (typical d50 value of
1-10 .mu.m) and/or molybdenum disulfide (MoS.sub.2, typical d50
value of 0.9-30 .mu.m).
[0040] In the various lubricants and/or methods discussed above and
herein, the lubricant can be more easily removed from a surface
(e.g. workpiece or human skin) in contact with the lubricant after
use compared to traditional solid film lubricants such as those
containing graphite and/or molybdenum disulfide which leave
difficult to remove residues.
DETAILED DESCRIPTION
Definitions
[0041] An "oleaginous" cell is a cell capable of producing at least
20% lipid by dry cell weight, naturally or through recombinant or
classical strain improvement. An "oleaginous microbe" or
"oleaginous microorganism" is a unicellular microbe, including a
microalga that is oleaginous. An oleaginous cell also encompasses a
cell that has had some or all of its lipid or other content
removed, and both live and dead cells. An "oleaginous microbial
biomass" may contain cells and/or intracellular contents as well as
extracellular material. Extracellular material includes, but is not
limited to, compounds secreted by a cell.
[0042] "Microalgae" refers to eukaryotic microbial organisms that
contain a chloroplast or other plastid, and optionally that are
capable of performing photosynthesis, or a prokaryotic microbial
organism capable of performing photosynthesis. Microalgae include
obligate photoautotrophs, which cannot metabolize a fixed carbon
source as energy, as well as heterotrophs, which can live solely
off of a fixed carbon source. Microalgae include unicellular
organisms that separate from sister cells shortly after cell
division, such as Chlamydomonas, as well as microbes such as, for
example, Volvox, which is a simple multicellular photosynthetic
microbe of two distinct cell types. Microalgae include cells such
as Chlorella, Dunaliella, and Prototheca. Microalgae also include
other microbial photosynthetic organisms that exhibit cell-cell
adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae
also include obligate heterotrophic microorganisms that have lost
the ability to perform photosynthesis. Examples of obligate
heterotrophs include certain dinoflagellate algae species and
species of the genus Prototheca. Microalgae include those belonging
to the phylum Chlorophyta and in the class Trebouxiophyceae. Within
this class are included microalgae belonging to the order
Chlorellales, optionally the family Chlorellaceae, and optionally
the genus Prototheca, Auxenochlorella, Chlorella, or
Parachlorella.
[0043] "Microalgal extracts" refer to any cellular components that
are extracted from the cell or are secreted by the cells. The
extracts include those can be obtained by mechanical pressing of
the cells or by solvent extraction. Cellular components can
include, but are not limited to, microalgal oil, proteins,
carbohydrates, phospholipids, polysaccharides, macromolecules,
minerals, cell wall, trace elements, carotenoids, and sterols. In
some cases the extract is a polysaccharide that is secreted from a
cell into the extracellular environment and has lost any physical
association with the cells. In other cases the polysaccharide
remain associated with the cell wall. Polysaccharides are typically
polymers of monosaccharide units and have high molecular weights,
usually with an average of 2 million Daltons or greater, although
fragments can be smaller in size.
[0044] "Microalgal oils" or "cell oils" refer to lipid components
produced by microalgal cells such as triglycerides.
[0045] "Modified microalgal extracts" refer to extracts that are
chemically or enzymatically modified. For example, triglyceride
extracts can be converted to fatty acid alkyl esters (e.g. fatty
acid methyl esters) by transesterification.
[0046] "Microalgal biomass," "algal biomass" or "biomass" refers to
material produced by growth and/or propagation of microalgal cells.
Biomass may contain cells and/or intracellular contents as well as
extracellular material. Extracellular material includes, but is not
limited to, compounds secreted by a cell.
[0047] "Floor sweep ingredient" refers to an ingredient
conventionally used in floor sweep compositions that is not
physically or chemically incompatible with the microalgal
components described herein. "Floor sweep ingredients" include,
without limitation, absorbents, abrasives, binders, vegetable oils,
petroleum derived oils, petroleum derivatives, antimicrobial
agents, bulking agents, and chemical additives. Such "floor sweep
ingredients" are known in the art.
[0048] "Metalworking" refers to cutting, grinding, punching, or
forming of metal. Metal forming includes any process that is
designed to alter the shape of metal while minimizing production of
small metal fragments (chips). These processes include but are not
limited to forging; extrusion; rod, wire or tube drawing; rolling;
and sheet forming. Examples of forging are such operations as
open-die forging, cogging, closed die forging, coining, nosing,
upsetting, heading, piercing, hobbing, roll forging, orbital
forging, ring rolling, rotary swaging of bars and tubes, and radial
forging. Examples of rolling are flat rolling or shape rolling.
Examples of sheet forming are blanking, piercing, press bending,
deep drawing, stamping, stretch forming, spinning, hydroforming,
rubber-pad forming, shallow recessing, explosive forming, dimpling,
roll forming, or flanging.
[0049] "Metalworking fluid ingredient" refers to an ingredient
conventionally used in metalworking fluid compositions that is not
physically or chemically incompatible with the microalgal
components described herein. "Metalworking fluid ingredients"
include, without limitation, antifoaming agents, antimicrobial
agents, binders, biocides, bacteriocides, fungicides, buffering
agents, chemical additives, pH adjusters, emulsifiers, lubricity
agents, vegetable oils, petroleum derived oils, petroleum
derivatives, corrosion inhibitors, extreme pressure additives,
defoamers, alkaline reserves, antimisting agents, couplers,
odorants, surfactants, humectants, thickeners, chelating agents,
and dyes. Such "metalworking fluid ingredients" are known in the
art.
[0050] "Dry weight" or "dry cell weight" refer to weight as
determined in the relative absence of water. For example, reference
to a component of microalgal biomass as comprising a specified
percentage by dry weight means that the percentage is calculated
based on the weight of the biomass after all or substantially all
water has been removed.
[0051] "Exogenous gene" refers to a nucleic acid transformed into a
cell. A transformed cell may be referred to as a recombinant cell,
into which additional exogenous gene(s) may be introduced. The
exogenous gene may be from a different species (and so
heterologous), or from the same species (and so homologous)
relative to the cell being transformed. In the case of a homologous
gene, it occupies a different location in the genome of the cell
relative to the endogenous copy of the gene. The exogenous gene may
be present in more than one copy in the cell. The exogenous gene
may be maintained in a cell as an insertion into the genome or as
an episomal molecule.
[0052] "Exogenously provided" describes a molecule provided to the
culture media of a cell culture.
[0053] "Fixed carbon source" means molecule(s) containing carbon,
preferably organic, that are present at ambient temperature and
pressure in solid or liquid form.
[0054] "Fatty acid profile" refers to the distribution of different
carbon chain lengths and saturation levels of fatty acid moieties
in a particular sample of biomass or oil. "Triglycerides" are
lipids where three fatty acid moieties are attached to a glycerol
moiety. A sample could contain lipids in which approximately 60% of
the fatty acid moieties is C18:1, 20% is C18:0, 15% is C16:0, and
5% is C14:0. In cases in which a carbon length is referenced
generically, such as "C18", such reference can include any amount
of saturation; for example, microalgal biomass that contains 20%
lipid as C18 can include C18:0, C18:1, C18:2, and the like, in
equal or varying amounts, the sum of which constitute 20% of the
biomass.
[0055] "Lipids" are a class of molecules that are soluble in
nonpolar solvents (such as ether and hexane) and are relatively or
completely insoluble in water. Lipid molecules have these
properties because they consist largely of long hydrocarbon tails
which are hydrophobic in nature. Examples of lipids include fatty
acids (saturated and unsaturated); glycerides or glycerolipids
(such as monoglycerides, diglycerides, triglycerides or neutral
fats, and phosphoglycerides or glycerophospholipids); nonglycerides
(sphingolipids, tocopherols, tocotrienols, sterol lipids including
cholesterol and steroid hormones, prenol lipids including
terpenoids, fatty alcohols, waxes, and polyketides); and complex
lipid derivatives (sugar-linked lipids, or glycolipids, and
protein-linked lipids).
[0056] "Homogenate" means biomass that has been physically
disrupted.
[0057] "Homogenize" means to blend two or more substances into a
homogenous or uniform mixture. In some embodiments, a homogenate is
created. In other embodiments, the biomass is predominantly intact,
but homogeneously distributed throughout the mixture.
[0058] "Predominantly intact cells" refers to a population of cells
which comprise more than 50%, 75%, or 90% intact cells. "Intact"
refers to the physical continuity of the cellular membrane
enclosing the intracellular components of the cell and means that
the cellular membrane has not been disrupted in any manner that
would release the intracellular components of the cell to an extent
that exceeds the permeability of the cellular membrane under
conventional culture conditions or those culture conditions
described herein.
[0059] "Predominantly lysed cells" refers to a population of cells
which comprise at least 75%, 55%, or 90% lysed cells.
[0060] "Delipidated cells" refers to a population of cells where
oil has been extracted from the cells, such that the extracted oil
is not in physical contact with the cells. In some embodiments, 50%
to 95% by weight of oil has been extracted from the cells. In some
embodiments, 5% to 30% by weight of oil remains in the delipidated
cells. In some embodiments, 10% to 15% by weight of oil remains in
the delipidated cells.
[0061] Reference to proportions by volume, i.e., "v/v," means the
ratio of the volume of one substance or composition to the volume
of a second substance or composition. For example, reference to a
composition that comprises 5% v/v microalgal oil and at least one
other ingredient means that 5% of the composition's volume is
composed of microalgal oil; e.g., a composition having a volume of
100 mm.sup.3 would contain 5 mm.sup.3 of microalgal oil and 95
mm.sup.3 of other constituents.
[0062] Reference to proportions by weight, i.e., "w/w," means the
ratio of the weight of one substance or composition to the weight
of a second substance or composition. For example, reference to a
composition that comprises 5% w/w microalgal biomass and at least
one other ingredient means that 5% of the composition is composed
of microalgal biomass; e.g., a 100 g composition would contain 5 g
of microalgal biomass and 95 g of other constituents.
Microalgal Cells and Extracts
[0063] The microalgal cells can be prepared and heterotrophically
cultured according to methods such as those described in
WO2008/151149, WO2010/063031, WO2010/045368, WO2010/063032,
WO2011/150411, WO2013/158938, 61/923,327 filed Jan. 3, 2014,
PCT/US2014/037898 filed May 13, 2014, and in U.S. Pat. No.
8,557,249. The microalgal cells can be wild type cells or can be
modified by genetic engineering and/or classical mutagenesis to
alter their fatty acid profile and/or lipid productivity or other
physical properties such as color.
[0064] In some embodiments, the cell wall of the microalgae must be
disrupted during the use of the industrial product in order to
release the active components. Hence, in some embodiments having
strains of microalgae with cell walls susceptible to disruption are
preferred.
[0065] In particular embodiments, the wild-type or genetically
engineered microalgae comprise cells that are at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, or at least 80% or
more oil by dry weight. Preferred organisms grow heterotrophically
(on sugars in the absence of light).
[0066] In some embodiments, the microalgae is from the genus
Chlorella. Chlorella is a genus of single-celled green algae,
belonging to the phylum Chlorophyta. Chlorella cells are generally
spherical in shape, about 2 to 10 .mu.m in diameter, and lack
flagella. Some species of Chlorella are naturally heterotrophic. In
some embodiments, the microalgae is Chlorella (auexnochlorella)
protothecoides, Chlorella ellipsoidea, Chlorella minutissima,
Chlorella zofinienesi, Chlorella luteoviridis, Chlorella kessleri,
Chlorella sorokiniana, Chlorella fusca var. vacuolata Chlorella
sp., Chlorella cf. minutissima or Chlorella emersonii. Other
species of Chlorella those selected from the group consisting of
anitrata, Antarctica, aureoviridis, candida, capsulate, desiccate,
ellipsoidea (including strain CCAP 211/42), emersonii, fusca
(including var. vacuolata), glucotropha, infusionum (including var.
actophila and var. auxenophila), kessleri (including any of UTEX
strains 397,2229,398), lobophora (including strain SAG 37.88),
luteoviridis (including strain SAG 2203 and var. aureoviridis and
lutescens), miniata, cf. minutissima, minutissima (including UTEX
strain 2341), mutabilis, nocturna, ovalis, parva, photophila,
pringsheimii, protothecoides (including any of UTEX strains 1806,
411, 264, 256, 255, 250, 249, 31, 29, 25 or CCAP 211/8D, or CCAP
211/17 and var. acidicola), regularis (including var. minima, and
umbricata), reisiglii (including strain CCP 11/8), saccharophila
(including strain CCAP 211/31, CCAP 211/32 and var. ellipsoidea),
salina, simplex, sorokiniana (including strain SAG 211.40B), sp.
(including UTEX strain 2068 and CCAP 211/92), sphaerica,
stigmatophora, trebouxioides, vanniellii, vulgaris (including
strains CCAP 211/11K, CCAP 211/80 and f. tertia and var.
autotrophica, viridis, vulgaris, vulgaris f. tertia, vulgaris f.
viridis), xanthella, and zofingiensis.
[0067] In addition to Chlorella, other genera of microalgae can
also be used in the methods and compositions provided herein. In
some embodiments, the microalgae is a species selected from the
group consisting Parachlorella kessleri, Parachlorella
beijerinckii, Neochloris oleabundans, Bracteacoccus, including B.
grandis, B. cinnabarinas, and B. aerius, Bracteococcus sp. or
Scenedesmus rebescens. Other nonlimiting examples of microalgae
species include those species from the group of species and genera
consisting of Achnanthes orientalis; Agmenellum; Amphiprora
hyaline; Amphora, including A. coffeiformis including A.c. linea,
A.c. punctata, A.c. taylori, A.c. tenuis, A.c. delicatissima, A.c.
delicatissima capitata; Anabaena; Ankistrodesmus, including A.
falcatus; Boekelovia hooglandii; Borodinella; Botryococcus braunii,
including B. sudeticus; Bracteoccocus, including B. aerius, B.
grandis, B. cinnabarinas, B. minor, and B. medionucleatus;
Carteria; Chaetoceros, including C. gracilis, C. muelleri, and C.
muelleri subsalsum; Chlorococcum, including C. infusionum;
Chlorogonium; Chroomonas; Chrysosphaera; Cricosphaera;
Crypthecodinium cohnii; Cryptomonas; Cyclotella, including C.
cryptica and C. meneghiniana; Dunaliella, including D. bardawil, D.
bioculata, D. granulate, D. maritime, D. minuta, D. parva, D.
peircei, D. primolecta, D. salina, D. terricola, D. tertiolecta,
and D. viridis; Eremosphaera, including E. viridis; Ellipsoidon;
Euglena; Franceia; Fragilaria, including F. crotonensis; Gleocapsa;
Gloeothamnion; Hymenomonas; Isochrysis, including I. aff. galbana
and I. galbana; Lepocinclis; Micractinium (including UTEX LB 2614);
Monoraphidium, including M. minutum; Monoraphidium; Nannochloris;
Nannochloropsis, including N. salina; Navicula, including N.
acceptata, N. biskanterae, N. pseudotenelloides, N. pelliculosa,
and N. saprophila; Neochloris oleabundans; Nephrochloris;
Nephroselmis; Nitschia communis; Nitzschia, including N.
alexandrina, N. communis, N. dissipata, N. frustulum, N.
hantzschiana, N. inconspicua, N. intermedia, N. microcephala, N.
pusilla, N. pusilla elliptica, N. pusilla monoensis, and N.
quadrangular; Ochromonas; Oocystis, including O. parva and O.
pusilla; Oscillatoria, including O. limnetica and O. subbrevis;
Parachlorella, including P. beijerinckii (including strain SAG
2046) and P. kessleri (including any of SAG strains 11.80, 14.82,
21.11H9); Pascheria, including P. acidophila; Pavlova; Phagus;
Phormidium; Platymonas; Pleurochrysis, including P. carterae and P.
dentate; Prototheca, including P. stagnora (including UTEX 327), P.
portoricensis, and P. moriformis (including UTEX strains 1441,
1435, 1436, 1437, 1439); Pseudochlorella aquatica; Pyramimonas;
Pyrobotrys; Rhodococcus opacus; Sarcinoid chrysophyte; Scenedesmus,
including S. armatus and S. rubescens; Schizochytrium; Spirogyra;
Spirulina platensis; Stichococcus; Synechococcus; Tetraedron;
Tetraselmis, including T. suecica; Thalassiosira weissflogii; and
Viridiella fridericiana.
Media and Culture Conditions for Microalgae
[0068] Microalgae are cultured in liquid media to propagate
biomass. Microalgal species are grown in a medium containing a
fixed carbon and/or fixed nitrogen source in the absence of light.
Such growth is known as heterotrophic growth. For some species of
microalgae, for example, heterotrophic growth for extended periods
of time such as 10 to 15 or more days under limited nitrogen
conditions results accumulation of high lipid content in cells.
[0069] Microalgal culture media typically contains components such
as a fixed carbon source (discussed below), a fixed nitrogen source
(such as protein, soybean meal, yeast extract, cornsteep liquor,
ammonia (pure or in salt form), nitrate, or nitrate salt), trace
elements (for example, zinc, boron, cobalt, copper, manganese, and
molybdenum in, e.g., the respective forms of ZnCl.sub.2,
H.sub.3BO.sub.3, CoCl.sub.2.6H.sub.2O, CuCl.sub.2.2H.sub.2O,
MnCl.sub.2.4H.sub.2O and
(NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O), optionally a buffer
for pH maintenance, and phosphate (a source of phosphorous; other
phosphate salts can be used). Other components include salts such
as sodium chloride, particularly for seawater microalgae.
[0070] In a particular example, a medium suitable for culturing
Chlorella protothecoides comprises Proteose Medium. This medium is
suitable for axenic cultures, and a 1 L volume of the medium (pH
.about.6.8) can be prepared by addition of 1 g of proteose peptone
to 1 liter of Bristol Medium. Bristol medium comprises 2.94 mM
NaNO.sub.3, 0.17 mM CaCl.sub.2.2H.sub.2O, 0.3 mM
MgSO.sub.4.7H.sub.2O, 0.43 mM, 1.29 mM KH.sub.2PO.sub.4, and 1.43
mM NaCl in an aqueous solution. For 1.5% agar medium, 15 g of agar
can be added to 1 L of the solution. The solution is covered and
autoclaved, and then stored at a refrigerated temperature prior to
use. Other methods for the growth and propagation of Chlorella
protothecoides to high oil levels as a percentage of dry weight
have been described (see for example Miao and Wu, J. Biotechnology,
2004, 11:85-93 and Miao and Wu, Biosource Technology (2006)
97:841-846 (demonstrating fermentation methods for obtaining 55%
oil dry cell weight)). High oil algae can typically be generated by
increasing the length of a fermentation while providing an excess
of carbon source under nitrogen limitation.
[0071] Solid and liquid growth media are generally available from a
wide variety of sources, and instructions for the preparation of
particular media that is suitable for a wide variety of strains of
microorganisms can be found, for example, online at a site
maintained by the University of Texas at Austin for its culture
collection of algae (UTEX). For example, various fresh water media
include 1/2, 1/3, 1/5, 1.times., 2/3, 2.times.CHEV Diatom Medium;
1:1 DYIII/PEA+Gr+; Ag Diatom Medium; Allen Medium; BG11-1 Medium;
Bold 1NV and 3N Medium; Botryococcus Medium; Bristol Medium; Chu's
Medium; CR1, CR1-S, and CR1+ Diatom Medium; Cyanidium Medium;
Cyanophycean Medium; Desmid Medium; DYIII Medium; Euglena Medium;
HEPES Medium; J Medium; Malt Medium; MES Medium; Modified Bold 3N
Medium; Modified COMBO Medium; N/20 Medium; Ochromonas Medium; P49
Medium; Polytomella Medium; Proteose Medium; Snow Algae Media; Soil
Extract Medium; Soilwater: BAR, GR-, GR-/NH4, GR+, GR+/NH4, PEA,
Peat, and VT Medium; Spirulina Medium; Tap Medium; Trebouxia
Medium; Volvocacean Medium; Volvocacean-3N Medium; Volvox Medium;
Volvox-Dextrose Medium; Waris Medium; and Waris+Soil Extract
Medium. Various Salt Water Media include: 1%, 5%, and 1.times.F/2
Medium; 1/2, 1.times., and 2.times. Erdschreiber's Medium; 1/2,
1/3, 1/4, 1/5, 1.times., 5/3, and 2.times. Soil+Seawater Medium;
1/4 ERD; 2/3 Enriched Seawater Medium; 20% Allen+80% ERD;
Artificial Seawater Medium; BG11-1+0.36% NaCl Medium; BG11-1+1%
NaCl Medium; Bold 1NV:Erdshreiber (1:1) and (4:1); Bristol-NaCl
Medium; Dasycladales Seawater Medium; 1/2 and 1.times. Enriched
Seawater Medium, including ES/10, ES/2, and ES/4; F/2+NH4; LDM
Medium; Modified 1.times. and 2.times.CHEV; Modified
2.times.CHEV+Soil; Modified Artificial Seawater Medium; Porphridium
Medium; and SS Diatom Medium.
[0072] Other suitable media for use with the methods provided
herein can be readily identified by consulting other organizations
that maintain cultures of microorganisms, such as SAG, CCAP, or
CCALA. SAG refers to the Culture Collection of Algae at the
University of Gottingen (Gottingen, Germany), CCAP refers to the
culture collection of algae and protozoa managed by the Scottish
Association for Marine Science (Scotland, United Kingdom), and
CCALA refers to the culture collection of algal laboratory at the
Institute of Botany (T ebo , Czech Republic).
[0073] Microorganisms useful in accordance with the methods of the
present disclosure are found in various locations and environments
throughout the world. As a consequence of their isolation from
other species and their resulting evolutionary divergence, the
particular growth medium for optimal growth and generation of oil
and/or lipid and/or protein from any particular species of microbe
can be difficult or impossible to predict, but those of skill in
the art can readily find appropriate media by routine testing in
view of the disclosure herein. In some cases, certain strains of
microorganisms may be unable to grow on a particular growth medium
because of the presence of some inhibitory component or the absence
of some essential nutritional requirement required by the
particular strain of microorganism. The examples below provide
exemplary methods of culturing various species of microalgae to
accumulate high levels of lipid as a percentage of dry cell
weight.
[0074] Suitable fixed carbon sources for use in the medium,
include, for example, glucose, fructose, sucrose, galactose,
xylose, mannose, rhamnose, arabinose, N-acetylglucosamine,
glycerol, floridoside, glucuronic acid, and/or acetate.
[0075] Process conditions can be adjusted to increase the
percentage weight of cells that is lipid. For example, in certain
embodiments, a microalgae is cultured in the presence of a limiting
concentration of one or more nutrients, such as, for example,
nitrogen, phosphorous, or sulfur, while providing an excess of a
fixed carbon source, such as glucose. Nitrogen limitation tends to
increase microbial lipid yield over microbial lipid yield in a
culture in which nitrogen is provided in excess. In particular
embodiments, the increase in lipid yield is at least about 10%,
50%, 100%, 200%, or 500%. The microbe can be cultured in the
presence of a limiting amount of a nutrient for a portion of the
total culture period or for the entire period. In some embodiments,
the nutrient concentration is cycled between a limiting
concentration and a non-limiting concentration at least twice
during the total culture period.
[0076] In a steady growth state, the cells accumulate oil but do
not undergo cell division. In one embodiment, the growth state is
maintained by continuing to provide all components of the original
growth media to the cells with the exception of a fixed nitrogen
source. Cultivating microalgal cells by feeding all nutrients
originally provided to the cells except a fixed nitrogen source,
such as through feeding the cells for an extended period of time,
results in a higher percentage of lipid by dry cell weight.
[0077] In other embodiments, high lipid biomass is generated by
feeding a fixed carbon source to the cells after all fixed nitrogen
has been consumed for extended periods of time, such as at least
one or two weeks. In some embodiments, cells are allowed to
accumulate oil in the presence of a fixed carbon source and in the
absence of a fixed nitrogen source for over 20 days. Microalgae
grown using conditions described herein or otherwise known in the
art can comprise at least about 20% lipid by dry weight, and often
comprise 35%, 45%, 55%, 65%, and even 75% or more lipid by dry
weight. Percentage of dry cell weight as lipid in microbial lipid
production can therefore be improved by holding cells in a
heterotrophic growth state in which they consume carbon and
accumulate oil but do not undergo cell division.
[0078] Organic nitrogen sources have been used in microbial
cultures since the early 1900s. The use of organic nitrogen
sources, such as corn steep liquor was popularized with the
production of penicillin from mold. Researchers found that the
inclusion of corn steep liquor in the culture medium increased the
growth of the microorganism and resulted in an increased yield in
products (such as penicillin). An analysis of corn steep liquor
determined that it was a rich source of nitrogen and also vitamins
such as B-complex vitamins, riboflavin panthothenic acid, niacin,
inositol and nutrient minerals such as calcium, iron, magnesium,
phosphorus and potassium (Ligget and Koffler, Bacteriological
Reviews (1948);12(4): 297-311). Organic nitrogen sources, such as
corn steep liquor, have been used in fermentation media for yeasts,
bacteria, fungi and other microorganisms. Non-limiting examples of
organic nitrogen sources are yeast extract, peptone, corn steep
liquor and corn steep powder. Non-limiting examples of preferred
inorganic nitrogen sources include, for example, and without
limitation, (NH.sub.4).sub.280.sub.4 and NH.sub.4OH. In one
embodiment, the culture media for contains only inorganic nitrogen
sources. In another embodiment, the culture media contains only
organic nitrogen sources. In yet another embodiment, the culture
media contains a mixture of organic and inorganic nitrogen
sources.
[0079] In some embodiments, a bioreactor or fermentor is used to
culture microalgal cells through the various phases of their
physiological cycle. As an example, an inoculum of lipid-producing
microalgal cells is introduced into the medium; there is a lag
period (lag phase) before the cells begin to propagate. Following
the lag period, the propagation rate increases steadily and enters
the log, or exponential, phase. The exponential phase is in turn
followed by a slowing of propagation due to decreases in nutrients
such as nitrogen, increases in toxic substances, and quorum sensing
mechanisms. After this slowing, propagation stops, and the cells
enter a stationary phase or steady growth state, depending on the
particular environment provided to the cells. For obtaining protein
rich biomass, the culture is typically harvested during or shortly
after then end of the exponential phase. For obtaining lipid rich
biomass, the culture is typically harvested well after then end of
the exponential phase, which may be terminated early by allowing
nitrogen or another key nutrient (other than carbon) to become
depleted, forcing the cells to convert the carbon sources, present
in excess, to lipid. Culture condition parameters can be
manipulated to optimize total oil production, the combination of
lipid species produced, and/or production of a specific oil.
[0080] Bioreactors offer many advantages for use in heterotrophic
growth and propagation methods. As will be appreciated, provisions
made to make light available to the cells in photosynthetic growth
methods are unnecessary when using a fixed-carbon source in the
heterotrophic growth and propagation methods described herein. To
produce biomass for use in industrial products, microalgae are
preferably fermented in large quantities in liquid, such as in
suspension cultures as an example. Bioreactors such as steel
fermentors (5000 liter, 10,000 liter, 40,000 liter, and higher) can
accommodate very large culture volumes. Bioreactors also typically
allow for the control of culture conditions such as temperature,
pH, oxygen tension, and carbon dioxide levels. For example,
bioreactors are typically configurable, for example, using ports
attached to tubing, to allow gaseous components, like oxygen or
nitrogen, to be bubbled through a liquid culture.
[0081] Bioreactors can be configured to flow culture media though
the bioreactor throughout the time period during which the
microalgae reproduce and increase in number. In some embodiments,
for example, media can be infused into the bioreactor after
inoculation but before the cells reach a desired density. In other
instances, a bioreactor is filled with culture media at the
beginning of a culture, and no more culture media is infused after
the culture is inoculated. In other words, the microalgal biomass
is cultured in an aqueous medium for a period of time during which
the microalgae reproduce and increase in number; however,
quantities of aqueous culture medium are not flowed through the
bioreactor throughout the time period. Thus in some embodiments,
aqueous culture medium is not flowed through the bioreactor after
inoculation.
[0082] Bioreactors equipped with devices such as spinning blades
and impellers, rocking mechanisms, stir bars, means for pressurized
gas infusion can be used to subject microalgal cultures to mixing.
Mixing may be continuous or intermittent. For example, in some
embodiments, a turbulent flow regime of gas entry and media entry
is not maintained for reproduction of microalgae until a desired
increase in number of said microalgae has been achieved.
[0083] As briefly mentioned above, bioreactors are often equipped
with various ports that, for example, allow the gas content of the
culture of microalgae to be manipulated. To illustrate, part of the
volume of a bioreactor can be gas rather than liquid, and the gas
inlets of the bioreactor to allow pumping of gases into the
bioreactor. Gases that can be beneficially pumped into a bioreactor
include air, air/CO.sub.2 mixtures, noble gases, such as argon, and
other gases. Bioreactors are typically equipped to enable the user
to control the rate of entry of a gas into the bioreactor. As noted
above, increasing gas flow into a bioreactor can be used to
increase mixing of the culture.
[0084] Increased gas flow affects the turbidity of the culture as
well. Turbulence can be achieved by placing a gas entry port below
the level of the aqueous culture media so that gas entering the
bioreactor bubbles to the surface of the culture. One or more gas
exit ports allow gas to escape, thereby preventing pressure buildup
in the bioreactor. Preferably a gas exit port leads to a "one-way"
valve that prevents contaminating microorganisms from entering the
bioreactor.
[0085] The specific examples of bioreactors, culture conditions,
and heterotrophic growth and propagation methods described herein
can be combined in any suitable manner to improve efficiencies of
microbial growth and lipid and/or protein production.
Concentration of Microalgae After Fermentation
[0086] Microalgal cultures generated according to the methods
described above yield microalgal biomass in fermentation media. To
prepare the biomass for use as a industrial product composition,
the biomass is concentrated, or harvested, from the fermentation
medium. At the point of harvesting the microalgal biomass from the
fermentation medium, the biomass comprises predominantly intact
cells suspended in an aqueous culture medium. To concentrate the
biomass, a dewatering step is performed. Dewatering or
concentrating refers to the separation of the biomass from
fermentation broth or other liquid medium and so is solid-liquid
separation. Thus, during dewatering, the culture medium is removed
from the biomass (for example, by draining the fermentation broth
through a filter that retains the biomass), or the biomass is
otherwise removed from the culture medium. Common processes for
dewatering include centrifugation, filtration, and the use of
mechanical pressure. These processes can be used individually or in
any combination.
[0087] Centrifugation involves the use of centrifugal force to
separate mixtures. During centrifugation, the more dense components
of the mixture migrate away from the axis of the centrifuge, while
the less dense components of the mixture migrate towards the axis.
By increasing the effective gravitational force (i.e., by
increasing the centrifugation speed), more dense material, such as
solids, separate from the less dense material, such as liquids, and
so separate out according to density. Centrifugation of biomass and
broth or other aqueous solution forms a concentrated paste
comprising the microalgal cells. Centrifugation does not remove
significant amounts of intracellular water. In fact, after
centrifugation, there may still be a substantial amount of surface
or free moisture in the biomass (e.g., upwards of 70%), so
centrifugation is not considered to be a drying step.
[0088] Filtration can also be used for dewatering. One example of
filtration that is suitable is tangential flow filtration (TFF),
also known as cross-flow filtration. Tangential flow filtration is
a separation technique that uses membrane systems and flow force to
separate solids from liquids. For an illustrative suitable
filtration method, see Geresh, Carb. Polym. 50; 183-189 (2002),
which describes the use of a MaxCell A/G Technologies 0.45 uM
hollow fiber filter. Also see, for example, Millipore Pellicon.RTM.
devices, used with 100 kD, 300 kD, 1000 kD (catalog number
P2C01MC01), 0.1 uM (catalog number P2VVPPV01), 0.22 uM (catalog
number P2GVPPV01), and 0.45 uM membranes (catalog number
P2HVMPV01). The retentate preferably does not pass through the
filter at a significant level, and the product in the retentate
preferably does not adhere to the filter material. TFF can also be
performed using hollow fiber filtration systems. Filters with a
pore size of at least about 0.1 micrometer, for example about 0.12,
0.14, 0.16, 0.18, 0.2, 0.22, 0.45, or at least about 0.65
micrometers, are suitable. Preferred pore sizes of TFF allow
solutes and debris in the fermentation broth to flow through, but
not microbial cells.
[0089] Dewatering can also be affected with mechanical pressure
directly applied to the biomass to separate the liquid fermentation
broth from the microbial biomass sufficient to dewater the biomass
but not to cause predominant lysis of cells. Mechanical pressure to
dewater microbial biomass can be applied using, for example, a belt
filter press. A belt filter press is a dewatering device that
applies mechanical pressure to a slurry (e.g., microbial biomass
taken directly from the fermentor or bioreactor) that is passed
between the two tensioned belts through a serpentine of decreasing
diameter rolls. The belt filter press can actually be divided into
three zones: the gravity zone, where free draining water/liquid is
drained by gravity through a porous belt; a wedge zone, where the
solids are prepared for pressure application; and a pressure zone,
where adjustable pressure is applied to the gravity drained
solids.
[0090] After concentration, microalgal biomass can be processed, as
described herein below, to produce vacuum-packed cake, algal
flakes, algal homogenate, algal powder, algal flour, or algal
oil.
Chemical Composition of Microalgal Biomass
[0091] The microalgal biomass generated by the culture methods
described herein comprises microalgal oil and/or protein as well as
other constituents generated by the microorganisms or incorporated
by the microorganisms from the culture medium during
fermentation.
[0092] Microalgal biomass with a high percentage of oil/lipid
accumulation by dry weight has been generated using different
methods of culture, including methods known in the art. Microalgal
biomass with a higher percentage of accumulated oil/lipid is useful
in accordance with the present disclosure. Chlorella vulgaris
cultures with up to 56.6% lipid by dry cell weight (DCW) in
stationary cultures grown under autotrophic conditions using high
iron (Fe) concentrations have been described (Li et al.,
Bioresource Technology 99(11):4717-22 (2008). Nanochloropsis sp.
and Chaetoceros calcitrans cultures with 60% lipid by DCW and 39.8%
lipid by DCW, respectively, grown in a photobioreactor under
nitrogen starvation conditions have also been described (Rodolfi et
al., Biotechnology & Bioengineering (2008)). Parietochloris
incise cultures with approximately 30% lipid by DCW when grown
phototropically and under low nitrogen conditions have been
described (Solovchenko et al., Journal of Applied Phycology
20:245-251 (2008). Chlorella protothecoides can produce up to 55%
lipid by DCW when grown under certain heterotrophic conditions with
nitrogen starvation (Miao and Wu, Bioresource Technology 97:841-846
(2006)). Other Chlorella species, including Chlorella emersonii,
Chlorella sorokiniana and Chlorella minutissima have been described
to have accumulated up to 63% oil by DCW when grown in stirred tank
bioreactors under low-nitrogen media conditions (Illman et al.,
Enzyme and Microbial Technology 27:631-635 (2000). Still higher
percent lipid by DCW has been reported, including 70% lipid in
Dumaliella tertiolecta cultures grown in increased NaCl conditions
(Takagi et al., Journal of Bioscience and Bioengineering 101(3):
223-226 (2006)) and 75% lipid in Botryococcus braunii cultures
(Banerjee et al., Critical Reviews in Biotechnology 22(3): 245-279
(2002)).
[0093] Heterotrophic growth results in relatively low chlorophyll
content (as compared to phototrophic systems such as open ponds or
closed photobioreactor systems). The reduced chlorophyll content
found in heterotrophically grown microalgae (e.g., Chlorella) also
reduces the green color in the biomass as compared to
phototrophically grown microalgae.
[0094] Oil rich microalgal biomass generated by the culture methods
described herein and useful in accordance with the present
disclosure comprises at least 10% microalgal oil by DCW (dry cell
weight). In some embodiments, the microalgal biomass comprises at
least 15%, 25%, 50%, 75% or at least 90% microalgal oil by DCW.
[0095] The microalgal oil of the biomass described herein (or
extracted from the biomass) can comprise glycerolipids with one or
more distinct fatty acid ester side chains. Glycerolipids are
comprised of a glycerol molecule esterified to one, two, or three
fatty acid molecules, which can be of varying lengths and have
varying degrees of saturation. Specific blends of algal oil can be
prepared either within a single species of algae, or by mixing
together the biomass (or algal oil) from two or more species of
microalgae.
[0096] Thus, the oil composition, i.e., the properties and
proportions of the fatty acid constituents of the glycerolipids,
can also be manipulated by combining biomass (or oil) from at least
two distinct species of microalgae. In some embodiments, at least
two of the distinct species of microalgae have different
glycerolipid profiles. The distinct species of microalgae can be
cultured together or separately as described herein, preferably
under heterotrophic conditions, to generate the respective oils.
Different species of microalgae can contain different percentages
of distinct fatty acid constituents in the cell's
glycerolipids.
[0097] In some embodiments, the microalgal oil is primarily
comprised of monounsaturated oil. In some cases, the algal oil is
at least 20% monounsaturated oil by weight. In various embodiments,
the algal oil is at least 25%, 50%, 75% or more monounsaturated oil
by weight or by volume. In some embodiments, the monounsaturated
oil is 18:1, 16:1, 14:1 or 12:1. In some embodiments, the
microalgal oil comprises at least 10%, 20%, 25%, or 50% or more
esterified oleic acid or esterified alpha-linolenic acid by weight
of by volume. In at least one embodiment, the algal oil comprises
less than 10%, less than 5%, less than 3%, less than 2%, or less
than 1% by weight or by volume, or is substantially free of,
esterified docosahexanoic acid (DHA (22:6)). For examples of
production of high DHA-containing microalgae, such as in
Crypthecodinium cohnii, see U.S. Pat. Nos. 7,252,979, 6,812,009 and
6,372,460.
[0098] Microalgal biomass generated by culture methods described
herein and useful in accordance to those embodiments of the present
disclosure relating to high protein typically comprises at least
30% protein by dry cell weight. In some embodiments, the microalgal
biomass comprises at least 40%, 50%, 75% or more protein by dry
cell weight. In some embodiments, the microalgal biomass comprises
from 30-75% protein by dry cell weight or from 40-60% protein by
dry cell weight. In some embodiments, the protein in the microalgal
biomass comprises at least 40% digestible crude protein. In other
embodiments, the protein in the microalgal biomass comprises at
least 50%, 60%, 70%, 80%, or at least 90% digestible crude protein.
In some embodiments, the protein in the microalgal biomass
comprises from 40-90% digestible crude protein, from 50-80%
digestible crude protein, or from 60-75% digestible crude
protein.
[0099] Microalgal biomass (and oil extracted therefrom), can also
include other constituents produced by the microalgae, or
incorporated into the biomass from the culture medium. These other
constituents can be present in varying amounts depending on the
culture conditions used and the species of microalgae (and, if
applicable, the extraction method used to recover microalgal oil
from the biomass). The other constituents can include, without
limitation, phospholipids (e.g., algal lecithin), carbohydrates,
soluble and insoluble fiber, glycoproteins, phytosterols (e.g.,
.beta.-sitosterol, campesterol, stigmasterol, ergosterol, and
brassicasterol), tocopherols, tocotrienols, carotenoids (e.g.,
.alpha.-carotene, .beta.-carotene, and lycopene), xanthophylls
(e.g., lutein, zeaxanthin, .alpha.-cryptoxanthin, and
.beta.-cryptoxanthin), proteins, polysaccharides (e.g., arabinose,
mannose, galactose, 6-methyl galactose and glucose) and various
organic or inorganic compounds (e.g., selenium). Microalgal sterols
may have anti-inflammatory, anti-matrix-breakdown, and improvement
of skin barrier effects when incorporated into a skincare product
such as described in section IV(f) and Example 26.
[0100] In some cases, the biomass comprises at least 10 ppm
selenium. In some cases, the biomass comprises at least 25% w/w
algal polysaccharide. In some cases, the biomass comprises at least
15% w/w algal glycoprotein. In some cases, the biomass comprises
between 0-115 mcg/g total carotenoids. In some cases, the biomass
comprises at least 0.5% algal phospholipids. In some cases, the oil
derived from the algal biomass contains at least 0.10 mg/g total
tocotrienols. In some cases, the oil derived from the algal biomass
contains between 0.125 mg/g to 0.35 mg/g total tocotrienols. In
some cases, the oil derived from the algal biomass contains at
least 5.0 mg/100 g total tocopherols. In some cases, the oil
derived from the algal biomass contains between 5.0 mg/100 g to 10
mg/100 g tocopherols.
Processing Microalgal Biomass
[0101] Drying the microalgal biomass, either predominantly intact
or in homogenate form, is advantageous to facilitate further
processing or for use of the biomass in the methods and
compositions described herein. Drying refers to the removal of free
or surface moisture/water from predominantly intact biomass or the
removal of surface water from a slurry of homogenized (e.g., by
micronization) biomass.
[0102] In one embodiment, the concentrated microalgal biomass is
drum dried to a flake form to produce algal flake, as described in
part A of this section. In another embodiment, the concentrated
microalgal biomass is spray or flash dried (i.e., subjected to a
pneumatic drying process) to form a powder containing predominantly
intact cells to produce algal powder, as described in part B of
this section. In another embodiment, oil is extracted from the
concentrated microalgal biomass to form algal oil, as described in
part C of this section.
A. Algal Flake
[0103] Algal flake is prepared from concentrated microalgal biomass
that is applied as a film to the surface of a rolling, heated drum.
The dried solids are then scraped off with a knife or blade,
resulting in a small flakes. U.S. Pat. No. 6,607,900 describes
drying microalgal biomass using a drum dryer without a prior
centrifugation (concentration) step, and such a process may be used
in accordance with the methods of the present disclosure.
[0104] Because the biomass may be exposed to high heat during the
drying process, it may be advantageous to add an antioxidant to the
biomass prior to drying. The addition of an antioxidant will not
only protect the biomass during drying, but also extend the
shelf-life of the dried microalgal biomass when stored. In a
preferred embodiment, an antioxidant is added to the microalgal
biomass prior to subsequent processing such as drying or
homogenization.
[0105] Additionally, if there is significant time between the
production of the dewatered microalgal biomass and subsequent
processing steps, it may be advantageous to pasteurize the biomass
prior to drying. Free fatty acids from lipases may form if there is
significant time between producing and drying the biomass. In one
embodiment, the pasteurized microalgal biomass is an algal
flake.
B. Algal Powder
[0106] Algal powder of the present disclosure is prepared from
concentrated microalgal biomass using a pneumatic or spray dryer
(see for example U.S. Pat. No. 6,372,460). In a spray dryer,
material in a liquid suspension is sprayed in a fine droplet
dispersion into a current of heated air. The entrained material is
rapidly dried and forms a dry powder. In some cases, a pulse
combustion dryer can also be used to achieve a powdery texture in
the final dried material. In other cases, a combination of spray
drying followed by the use of a fluid bed dryer is used to achieve
the optimal conditions for dried microbial biomass (see, for
example, U.S. Pat. No. 6,255,505). As an alternative, pneumatic
dryers can also be used in the production of algal powder.
Pneumatic dryers draw or entrain the material that is to be dried
in a stream of hot air. While the material is entrained in the hot
air, the moisture is rapidly removed. The dried material is then
separated from the moist air and the moist air is then recirculated
for further drying.
C. Algal Flour
[0107] Algal flour of the present disclosure is prepared from
concentrated microalgal biomass that has been mechanically lysed
and homogenized and the homogenate spray or flash dried (or dried
using another pneumatic drying system). The production of algal
flour requires that cells be lysed to release their oil and that
cell wall and intracellular components be micronized or reduced in
particle size to an average size of no more than 10 .mu.m. The
resulting oil, water, and micronized particles are emulsified such
that the oil does not separate from the dispersion prior to drying.
For example, a pressure disrupter can be used to pump a cell
containing slurry through a restricted orifice valve to lyse the
cells. High pressure (up to 1500 bar) is applied, followed by an
instant expansion through an exiting nozzle. Cell disruption is
accomplished by three different mechanisms: impingement on the
valve, high liquid shear in the orifice, and sudden pressure drop
upon discharge, causing an explosion of the cell. The method
releases intracellular molecules. A Niro (Niro Soavi GEA)
homogenizer (or any other high pressure homogenizer) can be used to
process cells to particles predominantly 0.2 to 5 microns in
length. Processing of algal biomass under high pressure
(approximately 1000 bar) typically lyses over 90% of the cells and
reduces particle size to less than 5 microns.
[0108] Alternatively, a ball mill can be used. In a ball mill,
cells are agitated in suspension with small abrasive particles,
such as beads. Cells break because of shear forces, grinding
between beads, and collisions with beads. The beads disrupt the
cells to release cellular contents. In one embodiment, algal
biomass is disrupted and formed into a stable emulsion using a
Dyno-mill ECM Ultra (CB Mills) ball mill. Cells can also be
disrupted by shear forces, such as with the use of blending (such
as with a high speed or Waring blender as examples), the french
press, or even centrifugation in case of weak cell walls, to
disrupt cells. A suitable ball mill including specifics of ball
size and blade is described in U.S. Pat. No. 5,330,913.
[0109] The immediate product of homogenization is a slurry of
particles smaller in size than the original cells that is suspended
in in oil and water. The particles represent cellular debris. The
oil and water are released by the cells. Additional water may be
contributed by aqueous media containing the cells before
homogenization. The particles are preferably in the form of a
micronized homogenate. If left to stand, some of the smaller
particles may coalesce. However, an even dispersion of small
particles can be preserved by seeding with a microcrystalline
stabilizer, such as microcrystalline cellulose.
[0110] To form the algal flour, the slurry is spray or flash dried,
removing water and leaving a dry power containing cellular debris
and oil. Although the oil content of the powder can be at least 10,
25 or 50% by weight of the dry powder, the powder can have a dry
rather than greasy feel and appearance (e.g., lacking visible oil)
and can also flow freely when shaken. Various flow agents
(including silica-derived products) can also be added. After
drying, the water or moisture content of the powder is typically
less than 10%, 5%, 3% or 1% by weight. Other dryers such as
pneumatic dryers or pulse combustion dryers can also be used to
produce algal flour.
[0111] The oil content of algal flour can vary depending on the
percent oil of the algal biomass. Algal flour can be produced from
algal biomass of varying oil content. In certain embodiments, the
algal flour is produced from algal biomass of the same oil content.
In other embodiments, the algal flour is produced from alglal
biomass of different oil content. In the latter case, algal biomass
of varying oil content can be combined and then the homogenization
step performed. In other embodiments, algal flour of varying oil
content is produced first and then blended together in various
proportions in order to achieve an algal flour product that
contains the final desired oil content. In a further embodiment,
algal biomass of different lipid profiles can be combined together
and then homogenized to produce algal flour. In another embodiment,
algal flour of different lipid profiles is produced first and then
blended together in various proportions in order to achieve an
algal flour product that contains the final desired lipid
profile.
D. Algal Oil
[0112] Algal oil can be separated from lysed biomass. The algal
biomass remaining after oil extraction is referred to as
delipidated meal, delipidated cells, or delipidated biomass.
Delipidated meal contains less oil by dry weight or volume than the
microalgae contained before extraction. Typically 50-90% of oil can
be extracted so that delipidated meal contains, for example, 10-50%
of the oil content of biomass before extraction.
[0113] In some embodiments, the algal oil is at least 50% w/w oleic
acid and contains less than 5% DHA. In some embodiments of the
method, the algal oil is at least 50% w/w oleic acid and contains
less than 0.5% DHA. In some embodiments of the method, the algal
oil is at least 50% w/w oleic acid and contains less than 5%
glycerolipid containing carbon chain length greater than 18. In
some cases, the algal cells from which the algal oil is obtained
comprise a mixture of cells from at least two distinct species of
microalgae. In some cases, at least two of the distinct species of
microalgae have been separately cultured. In at least one
embodiment, at least two of the distinct species of microalgae have
different glycerolipid profiles. In some cases, the algal cells are
cultured under heterotrophic conditions. In some cases, all of the
at least two distinct species of microalgae contain at least 10%,
or at least 15% oil by dry weight.
[0114] Microalgae containing lipids can be lysed to produce a
lysate. As detailed herein, the step of lysing a microorganism
(also referred to as cell lysis) can be achieved by any convenient
means, including heat-induced lysis, adding a base, adding an acid,
using enzymes such as proteases and polysaccharide degradation
enzymes such as amylases, using ultrasound, mechanical
pressure-based lysis, and lysis using osmotic shock. Each of these
methods for lysing a microorganism can be used as a single method
or in combination simultaneously or sequentially. The extent of
cell disruption can be observed by microscopic analysis. Using one
or more of the methods above, typically more than 70% cell breakage
is observed. Preferably, cell breakage is more than 80%, more
preferably more than 90% and most preferred about 100%.
Combining Microalgal Biomass or Materials Derived Therefrom with
Other Industrial Lubricant Ingredients
[0115] In one aspect, provided is a method of combining microalgal
biomass with at least one other metalworking fluid ingredient to
form a metalworking fluid composition.
[0116] In some cases, the metalworking fluid composition formed by
the combination of microalgal biomass comprises at least 1%, at
least 5%, at least 10%, at least 25%, or at least 50% w/w
microalgal biomass. In some embodiments, the oil of microalgal
biomass of the metalworking composition has a fatty acid profile of
at least 50%, at least 60%, at least 70%, at least 80%, at least
85%, at least 90%, or at least 95% oleic acid. In some cases, the
fatty acid profile has less than 6%, 5%, 4%, 3%, 2%, 1%, 0.9%,
0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01%
polyunsaturated fatty acids.
[0117] In some cases, the metalworking fluid composition formed by
the combination of microalgal oil comprises at least 1%, at least
5%, at least 10%, at least 25%, at least 50%, at least 70%, at
least 90%, or at least 99% w/w microalgal oil. In some embodiments,
metalworking fluid compositions formed as described herein comprise
at least 2%, at least 3%, at least 4%, at least 15%, at least 20%,
at least 30%, at least 35%, at least 40%, at least 45%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, or at least 95% w/w
microalgal oil. In some embodiments, the microalgal oil of the
metalworking composition has a fatty acid profile of at least 75%,
at least 80%, at least 85%, or at least 90% oleic acid. In some
cases, the fatty acid profile has less than 6%, 5%, 4%, 3%, 2%, 1%,
0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or
0.01% polyunsaturated fatty acids.
[0118] In some cases, the metalworking fluid composition formed by
the combination of microalgal fatty acid esters comprises at least
1%, at least 5%, at least 10%, at least 25%, at least 50%, at least
70%, at least 90%, or at least 99% w/w microalgal fatty acid
esters. In some embodiments, metalworking fluid compositions formed
as described herein comprise at least 2%, at least 3%, at least 4%,
at least 15%, at least 20%, at least 30%, at least 35%, at least
40%, at least 45%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% w/w microalgal fatty acid esters. In some
embodiments, the microalgal fatty acid esters of the metalworking
composition has a fatty acid profile of at least 75%, at least 80%,
at least 85%, or at least 90% oleic acid. In some cases, the fatty
acid profile has less than 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%,
0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01%
polyunsaturated fatty acids.
[0119] In some cases, the metalworking fluid comprises
predominantly intact microalgal cells. In some cases, the
composition comprises at least 50% intact cells, or at least 60%,
at least 70%, or at least 80% intact cells, or at least 90% intact
cells.
A. Substitution of Algal Biomass, Algal Oil, and Algal Oil
Derivatives in Industrial Lubricants
[0120] In some cases, microalgal biomass can be substituted for
other components that would otherwise be conventionally included in
a metalworking fluid product. In at least one embodiment, the
metalworking fluid composition formed by the methods of the
invention is free of oil other than microalgal oil contributed by
the microalgal biomass and entrapped therein.
[0121] In various embodiments, microalgal biomass can be
substituted for all or a portion of conventional metalworking fluid
ingredient such as lubricants, emulsifiers, and the like, to the
extent that the components of the microalgal biomass replace the
corresponding conventional components in like kind, or adequately
substitute for the conventional components to impart the desired
characteristics to the metalworking fluid composition.
B. Other Metalworking Fluid Ingredients
[0122] Microalgal biomass and microalgal oil and oil derivatives
are combined with at least one other metalworking fluid ingredients
in methods of the present disclosure to form metalworking fluid
compositions. The at least one other metalworking fluid ingredient
can be selected from conventional metalworking fluid ingredients
suitable for use with the microalgal biomass or microalgal oil with
regard to the intended use of the composition. Such other
metalworking fluid ingredients include, without limitation,
antifoaming agents, antimicrobial agents, binders, biocides,
bacteriocides, fungicides, chelating agents, chemical additives, pH
adjusters, emulsifiers, lubricity agents, vegetable oils, petroleum
derived oils, petroleum derivatives, corrosion inhibitors, extreme
pressure additives, defoamers, alkaline reserves, antimisting
agents, couplers, odorants, surfactants, humectants, rheology
modifiers, dyes, and other additives.
[0123] Specific examples of other metalworking fluid ingredients
are described below. Any one or more of these can be optionally
combined with microalgal biomass, microalgal oil, or derivatives of
microalgal oil in accordance with the present disclosure to form a
metalworking fluid composition. The ingredients described below are
categorized by their benefit or their postulated mode of action in
a metalworking fluid. However, it is to be understood that these
ingredients can in some instances provide more than one function
and/or operate via more than one mode of action. Therefore,
classifications herein are made for the sake of convenience and are
not intended to limit the ingredient to that particular application
or applications listed.
[0124] An effective amount of an anti-foaming agent can optionally
be added to the compositions of the present disclosure, preferably
from about 0.1% to about 3%, more preferably from about 0.5% to
about 1%, of the composition. The anti-foaming agent reduces or
controls the foaming properties of the fluid, e.g., such agents
contribute to an acceptable low level of foam. The exact amount of
anti-foaming agent to be used in the compositions will depend on
the particular anti-foaming agent utilized since such agents vary
widely in potency.
[0125] Anti-foaming agents, including but not limited to, are
silicones, waxes, calcium nitrates, and calcium acetate.
[0126] The metalworking compositions of the present disclosure may
contain an effective amount of one or more antimicrobial agents,
such that the resultant composition is safe and effective for
preventing, prohibiting, or retarding microbial growth in the
metalworking fluid. The compositions preferably contain from or
about 0.005% to or about 6%, more preferably 0.01% to or about 3%
antimicrobial agent. Antimicrobial agents may be broad spectrum or
may target specific types of bacteria or fungus. The exact amount
of antimicrobial agent to be used in the compositions will depend
on the particular antimicrobial agent utilized since such agents
vary widely in potency.
[0127] Antimicrobial agents may include but are not limited to
1,2-Benzisothiazolin-3-one, sodium omadine, phenolics,
p-chloro-m-cresol, halogen substituted carbamates, isothiazolone
derivatives, bromonitriles dinitromorpholines, amphotericin,
triazine, BIT, MIT, potassium sorbate, sodium benzoate, and include
those marketed under trade st, pyridinethione, polyquat, IPBC, OIT,
CTAC, CMIT, glutaraldehyde, Bronopol, DBPNA, Grotan (Troy), BIOBAN
(Dow).
[0128] The metalworking compositions of the present disclosure may
contain an effective amount of one or more chelating agents, such
that the resultant composition is effective for complexing with
water hardness ions to stabilize the fluid. The compositions
preferably contain from or about 0.005% to or about 5%, more
preferably 0.01% to or about 2% chelating agent.
[0129] Chelating agents may include but are not limited to sodium
ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid,
phosphonates, and gluconates.
[0130] The metalworking compositions of the present disclosure may
contain an effective amount of one or more pH adjusters, such that
the resultant composition is effective for maintaining desired pH.
The compositions preferably contain from or about 0.005% to or
about 5%, more preferably 0.01% to or about 2% pH adjuster. The
exact amount of pH agent to be used in the compositions will depend
on the particular pH agent utilized since such agents vary widely
in potency.
[0131] pH adjusters may include but are not limited to alkali
hydroxides, sodium hydroxide, potassium hydroxide, triethanolamine,
triethylamine, and alkanolamines.
[0132] The metalworking compositions of the present disclosure may
contain an effective amount of one or more emulsifiers, such that
the resultant composition maintains lubricant in suspension. The
compositions preferably contain from or about 0.5% to or about 15%,
more preferably 1% to or about 10% emulsifier. The exact amount of
emulsifier to be used in the compositions will depend on the
particular agent utilized since such agents vary widely in
potency.
[0133] Emulsifiers may include but are not limited to sodium
sulfonate, fatty acid soaps, nonionic ethoxylates, synthetic
sulfonates, fatty acid amines, and amphoterics.
[0134] The metalworking compositions of the present disclosure may
contain an effective amount of one or more lubricity agents, such
that the resultant composition provides or increases film strength
or a boundary effective for preventing metal-on-metal contact. The
compositions preferably contain from or about 0.5% to or about 90%
lubricity agent.
[0135] Lubricity agents may include but are not limited to
napthenic oils, paraffinc oils, fatty acid esters, high molecular
weight esters, glycol esters, ethylene oxide copolymers,
polypropylene oxide copolymers, naturally occurring triglycerides,
graphite, graphite fluoride, molybdenum disulfide, tungsten
disulfide, tin sulfide, and boron nitride.
[0136] The metalworking compositions of the present disclosure may
contain an effective amount of one or more corrosion inhibitors,
such that the resultant composition is effective for preventing
oxidation of metal parts and tools that come in contact with the
composition. The compositions preferably contain from or about
0.005% to or about 5% of a corrosion inhibitor. We also found that
metalworking compositions comprising microalgal biomas inhibited
corrosion)
[0137] Corrosion inhibitors may include but are not limited to
include amine carboxylates, amine dicarboxylates, amine
tricarboxylates, amine alcohols, boramides, arylsulfonamido acids,
sodium borate, sodium molybdate, sodium metasilicates, succinic
acid metasilicates, succinic acid derivates, tolyl and
benzotriazoles, and thiadiazoles.
[0138] The metalworking compositions of the present disclosure may
contain an effective amount of one or more extreme pressure
additives, such that the resultant composition is effective for
preventing welding of metal. The compositions preferably contain
from or about 5% to or about 30% extreme pressure additives.
[0139] Extreme pressure additives may include but are not limited
to sulfurized hydrocarbons, sulfurized fatty acid esters,
halogenated paraffins, halogenated waxes, halogenated fats,
halogenated esters, and phosphate esters.
[0140] The metalworking compositions of the present disclosure may
contain an effective amount of one or more rheology modifiers, such
that the resultant composition demonstrates viscosity and
flowability effective the intended use of the composition. The
compositions preferably contain from or about 0.005% to or about
5%, more preferably 0.01% to or about 2% rheology modifiers.
[0141] Rheology modifiers may include but are not limited to
hydroxyethyl cellulose, carboxymethyl cellulose, xanthan gum, guar
gum, starch, or polyanionic cellulose.
[0142] The metalworking compositions of the present disclosure may
contain an effective amount of one or more surfactants, such that
the resultant composition demonstrates effective wettability and
cleanability. The compositions preferably contain from or about
0.01% to or about 25%, more preferably 0.1% to or about 10%
surfactants.
[0143] Surfactants may include but are not limited to alkoxylated
alcohols alkoxylated nonylphenols.
C. Industrial Lubricant Compositions of Microalgal Biomass, Algal
Oil, and Algal Oil Derivatives
[0144] In one aspect, provided are metalworking compositions
comprising at least 1% w/w microalgal biomass and/or microalgal oil
and/or microalgal oil derivative. In some embodiments, the
compositions comprise at least 2%, at least 5%, at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% microalgal biomass and/or
microalgal oil and/or microalgal oil derivative. The remainder of a
metalworking fluid composition in accordance with the present
disclosure comprises water or other conventional ingredients,
including those identified herein.
[0145] Metalworking fluid compositions can be in the form of a
concentrated fluid. In other cases, the metalworking fluid
compositions of the present disclosure are in a diluted form.
[0146] The microalgal biomass useful in the metalworking fluid
compositions of the present disclosure can be derived from one or
more species of microalgae cultured and/or genetically engineered
as described herein.
[0147] In some embodiments, metalworking fluid compositions
comprise at least 1% w/w microalgal oil, or a greater percentage as
described above. The microalgal oil is derived from cultures of
microalgae grown under heterotrophic conditions or those comprising
at least 10% oil by dry cell weight, as described herein. In some
cases, the microalgae can be genetically engineered.
[0148] In one embodiment, provided is a method of preparing a
lubricant composition comprising (i) culturing a population of
microalgae under conditions to generate microalgal biomass
comprising at least 50% microalgal oil by dry weight, (ii)
harvesting the biomass from the microalgal culture, (iii)
performing one or more optional processing steps, e.g., drying the
biomass or extracting oil from the biomass, (iv) combining the
biomass with at least one other lubricant ingredient to form a
lubricant.
Floor Sweep Compositions
[0149] In use, floor sweep compositions are scattered over the
floor preliminary to the sweeping operation, to enable the
composition to pick up and hold dust, particulates fluid, or other
litter accumulated on the floor so that the floor may then be
cleanly swept by the action of the broom or other sweeping agent.
By thus causing the dust, particulates, fluid or litter to be
accumulated on the sweeping composition, the sweeping operation may
also be performed without the rising of dust under the action of
the broom.
[0150] Floor sweep compositions are conventionally comprised of
finely divided solid material and a moistening or wetting agent.
Solid carriers such as sawdust, rice hulls, oat hulls, corncobs and
sand have been used for years as a medium to which a wetting agent
adheres. Sand, when used, functions as both a carrier and abrading
cleaner, as well as a weighting compound to assure that the
sweeping composition will "hug" the floor. Variable proportions of
sand may be used, depending upon the age and the composition of the
floor being cleaned. For example, with newly finished floors, sand
in the composition is usually eliminated. However, as a floor gets
older and abraded, sand is used to make sure that the composition
effectively hugs the floor and causes slight abrasion to enhance
cleaning.
[0151] Conventional floor sweep compositions typically comprise a
petroleum-derived oil, such as a mineral oil or a bottoms residue
from petroleum refinement, as wetting agent that serves
additionally as a dust control agent. While often effective,
petroleum-derived oil presents a disadvantage in that oil-saturated
sweeping compound becomes an environmental pollutant, disposal of
which may often be difficult.
[0152] An unpleasant odor characteristic of petroleum-derived oil
is a further disadvantage of some conventional floor sweep
compositions.
[0153] Biologically-derived alternatives to petroleum-derived oil
wetting agents have been incorporated into floor sweep compositions
that demonstrate improved odor characteristics and ameliorate the
environmental pollutant disadvantage characteristic of floor sweep
compositions prepared with petroleum-derived oil. Some natural;
wetting agent alternatives include vegetable oils and water.
[0154] An further disadvantage of some conventional floor sweep
compositions comprising petroleum-derived oil, vegetable oil, or is
that upon storage, the oil wetting agent.
[0155] There is therefore a continuing need for development of
effective floor sweep compositions that avoid the inherent odor,
disposal, and leakage problems of an petroleum-based oil additive,
or at least reduce the petroleum-based oil content, but at the same
time, will still provide the effective dust control normally
associated with oil use.
[0156] In one aspect, provided is a method of combining microalgal
biomass with at least one other floor sweep ingredient to form a
floor sweep composition.
[0157] In some cases, the floor sweep composition formed by the
combination of microalgal biomass comprises at least 1%, at least
5%, at least 10%, at least 25%, at least 50%, at least 70%, or at
least 90% w/w microalgal biomass. In some embodiments, the oil of
microalgal biomass of the floor sweep composition has a fatty acid
profile of at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 85%, at least 90%, or at least 95% oleic
acid. In some embodiments, the oil of microalgal biomass of the
floor sweep composition has a fatty acid profile of at least 40%,
at least 50%, at least 60%, at least 70%, or at least 75% lauric
acid. In some cases, the fatty acid profile has less than 6%, 5%,
4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%,
0.1%, 0.05%, or 0.01% polyunsaturated fatty acids.
[0158] In some cases, the floor sweep composition formed by the
combination of microalgal biomass comprises at least 1%, at least
5%, at least 10%, at least 25%, at least 50%, at least 70%, or at
least 90% w/w delipidatated microalgal biomass.
[0159] In some cases, the floor sweep composition comprises
predominantly intact microalgal cells. In some cases, the floor
sweep composition comprises at least 50% intact cells, or at least
60%, at least 70%, or at least 80% intact cells, or at least 90%
intact cells.
[0160] In some cases, the floor sweep composition formed by the
combination of microalgal biomass comprises predominantly
delipidated microalgal meal. In some cases, the floor sweep
composition comprises at least 50%, or at least 60%, at least 70%,
or at least 80%, or at least 90% delipidated microalgal meal.
[0161] In some cases, the floor sweep composition formed by the
combination of microalgal biomass comprises a blend of delipidated
microalgal meal and intact microalgal cells. In some cases, the
floor sweep composition comprises a blend of equal parts
delipidated microalgal meal and intact microalgal cells.
A. Substitution of Algal Biomass, Algal Oil, and Algal Oil
Derivatives in Floor Sweep Products
[0162] In some cases, microalgal biomass can be substituted for
other components that would otherwise be conventionally included in
a floor sweep product. In at least one embodiment, the floor sweep
composition formed by the methods of the present disclosure is free
of oil other than microalgal oil contributed by the microalgal
biomass and entrapped therein.
[0163] In various embodiments, microalgal biomass can be
substituted for all or a portion of conventional floor sweep
ingredients such as absorbents, abrasives, carriers, and the like,
to the extent that the components of the microalgal biomass replace
the corresponding conventional components in like kind, or
adequately substitute for the conventional components to impart the
desired characteristics to the floor sweep composition.
[0164] In some cases, microalgal oil can be substituted for oils
conventionally used in floor sweep compositions. As described
herein, oils produced by microalgae can be tailored by culture
conditions or lipid pathway engineering to comprise particular
fatty acid components. Thus, the oils generated by the microalgae
the present disclosure can be used to replace conventional floor
sweep ingredients such as mineral oils, vegetable oils, and the
like. In at least one embodiment, the floor sweep composition
formed by the methods the present disclosure is free of oil other
than microalgal oil.
B. Other Floor Sweep Ingredients
[0165] Microalgal biomass and microalgal oil are combined with at
least one other floor sweep ingredient in methods the present
disclosure to form floor sweep compositions. The at least one other
floor sweep ingredient can be selected from conventional floor
sweep ingredients suitable for use with the microalgal biomass or
microalgal oil with regard to the intended use of the composition.
Such other floor sweep ingredients include, without limitation,
absorbents, abrasants, binders, antimicrobial agents, vegetable
oils, petroleum derived oils, odorants, dyes, weighting agents, and
other additives.
[0166] Specific examples of other floor sweep ingredients are
described below. Any one or more of these can be optionally
combined with microalgal biomass, microalgal oil, or derivatives in
accordance with the present disclosure to form a floor sweep
composition. The ingredients described below are categorized by
their benefit or their postulated mode of action in a floor sweep
composition. However, it is to be understood that these ingredients
can in some instances provide more than one function and/or operate
via more than one mode of action. Therefore, classifications herein
are made for the sake of convenience and are not intended to limit
the ingredient to that particular application or applications
listed.
[0167] An effective amount of one or more absorbent agent can
optionally be added to the compositions of the present disclosure,
preferably from about 1% to about 90%, more preferably from about
1% to about 70%, of the composition. The absorbent agent attracts
liquids or solid particles. The exact amount of absorbent agent to
be used in the compositions will depend on the particular absorbent
agent utilized since such agents vary widely in potency and vary in
selectivity.
[0168] Exemplary absorbent agents include without limitation ground
corncobs, soybean hulls, cellulose, sawdust, cotton fabric,
newspaper, superabsorbents, acrylate copolymers, calcium carbonate,
and calcium chloride.
[0169] An effective amount of one or more binding agent can
optionally be added to the compositions of the present disclosure,
preferably from about 1% to about 20% of the composition. The
binding agent binds. Binding agents may include vegetable oil,
soapstock, acid oil, glycerin, mineral oil, paraffin wax, and
rubber.
[0170] Exemplary binding agents may include water, vegetable oil,
soapstock, acid oil, glycerin, mineral oil, paraffin wax, rubber,
and processed tires.
[0171] An effective amount of one or more weighting agent can
optionally be added to the compositions of the present disclosure,
preferably from about 1% to about 20% of the composition. The
weighting agent adds mass to the composition and influences its
flow or spreading properties.
[0172] Exemplary weighting agents may include sand, silica,
volcanic ash, marble dust, limestone, and dyes.
[0173] The floor sweep compositions of the present disclosure may
contain an effective amount of one or more antimicrobial agents,
such that the resultant composition is safe and effective for
preventing, prohibiting, or retarding microbial growth in the floor
sweep. The compositions preferably contain from or about 0.005% to
or about 6%, more preferably 0.01% to or about 3% antimicrobial
agent. Antimicrobial agents may be broad spectrum or may target
specific types of bacteria or fungus. The exact amount of
antimicrobial agent to be used in the compositions will depend on
the particular antimicrobial agent utilized since such agents vary
widely in potency.
[0174] Antimicrobial agents may include but are not limited to
1,2-Benzisothiazolin-3-one, sodium omadine, phenolics,
p-chloro-m-cresol, halogen substituted carbamates, isothiazolone
derivatives, bromonitriles dinitromorpholines, amphotericin,
triazine, BIT, MIT, potassium sorbate, sodium benzoate, and include
those marketed under trade names Proxel GXL, pyridinethione,
polyquat, IPBC, OIT, CTAC, CMIT, glutaraldehyde, Bronopol, DBPNA,
Grotan (Troy), BIOBAN (Dow), such as marketed by Chantal
Pharmaceutical of Los Angeles, Calif. under the trade names ETHOCYN
and CYOCTOL, and 2-(5-ethoxy hept-1-yl)bicylo[3.3.0]octanone).
C. Floor Sweep Compositions of Microalgal Biomass, Algal Oil, and
Algal Oil Derivatives
[0175] In one aspect, provided are floor sweep compositions
comprising at least 1% w/w microalgal biomass and/or microalgal oil
and/or microalgal oil derivative. In some embodiments, the
compositions comprise at least 2%, at least 5%, at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% microalgal biomass and/or
microalgal oil and/or microalgal oil derivative. The remainder of a
floor sweep composition in accordance with the present disclosure
comprises water or other conventional ingredients, including those
identified herein.
[0176] In some embodiments, compositions of the present disclosure
comprise at least 1% w/w microalgal biomass, or a greater
percentage as described above. The microalgal biomass comprises at
least 10% microalgal oil by dry weight, and can include greater
amounts of microalgal oil as well as other constituents as
described herein.
[0177] The microalgal biomass useful in the floor sweep
compositions of the present disclosure can be derived from one or
more species of microalgae cultured and/or genetically engineered
as described herein.
[0178] In some embodiments, floor sweep compositions provided
herein comprise at least 1% w/w microalgal oil, or a greater
percentage as described above. The microalgal oil is derived from
cultures of microalgae grown under heterotrophic conditions or
those comprising at least 10% oil by dry cell weight, as described
herein. In some cases, the microalgae can be genetically
engineered.
[0179] The floor sweep compositions provided herein comprise at
least 1% w/w microalgal oil, or a greater percentage as described
above. The microalgal oil is derived from cultures of microalgae
grown under heterotrophic conditions or those comprising at least
10% oil by dry cell weight, as described herein. In some cases, the
microalgae can be genetically engineered.
[0180] In one aspect, the floor sweep compositions provides
advantages over other floor sweep compositions. For example, oil
based floor sweep compositions cannot be disposed of without
environmental restrictions and leave an oily residue sweeping.
Water-based sweeping compounds cannot be broadcast over an entire
floor area, but must be spread in a line and quickly swept up.
EXAMPLES
[0181] The following examples are offered to illustrate, but not to
limit, the claimed invention.
Example 1
[0182] Strains were prepared and grown heterotrophically as
described above and in WO2008/151149, WO2010/063031, WO2010/045368,
WO2010/063032, WO2011/150411, WO2013/158938, 61/923,327 filed Jan.
3, 2014, PCT/US2014/037898 filed May 13, 2014, and in U.S. Pat. No.
8,557,249. Sample IA refers to triglyceride oil from Chlorella
(Auxeochlorella) protothecoides cells (UTEX 250). Samples IB-IG are
oil isolated from various strains originating from Prototheca
moriformis (UTEX 1435) that were prepared and cultured to achieve
the indicated fatty acid profile. UTEX 250 and 1435 are available
from the University of Texas at Austin Culture Collection of
Algae.
TABLE-US-00001 TABLE I Oil properties Sample Assay IA IB (high IC
ID (high IE IF (low poly- IG Fatty Acid (UTEX 250) C10-C12)
(laurate) myristic) (SOS) unsaturates) (Oleic) Profile Units S106
S6207 S5223 S4845 S7586 S6697 S5587 C8:0 % 0.00 1.02 0.35 0.00 0.00
0.00 0.00 C10:0 % 0.08 40.45 18.18 0.04 0.03 0.03 0.01 C12:0 % 0.22
45.00 45.92 0.89 0.19 0.06 0.03 C14:0 % 1.29 4.00 12.92 56.94 0.47
0.35 0.41 C16:0 % 17.44 2.33 6.34 14.98 3.03 3.29 3.31 C18:0 % 1.66
0.27 0.51 0.68 56.75 2.87 2.22 C18:1 % 59.12 4.24 10.12 20.51 33.90
89.94 86.17 C18:2 % 15.17 1.62 3.32 4.26 1.94 1.03 5.50 C18:3 ALPHA
% 2.01 0.27 0.38 0.23 0.16 0.15 0.24 C20:0 % 0.25 0.02 0.06 0.06
1.65 0.25 0.26 DROPPING .degree. C. 10.5 22.2 27.2 2 0.3 MELTING
POINT (METTLER) AOCS Cc 18-80 CLOUD .degree. C. 12 17 29 -18 -19
POINT D97 POUR .degree. C. 10 15 27 -20 -21 POINT D97 IODINE VALUE
unit 85.6 8.8 18.7 27.7 81.6 85.6 OSI RANCIMAT hours 68.72 46.8
37.56 57.6 19.35 (110.degree. C.) AOCS Cd 12b-92 SMOKE POINT
.degree. C. 150 248 248 AOCS Cc 9a-48 SAPONIFICATION mg KOH/g 239.2
VALUE AOCS Cd 3-25 ALPHA mg/100 g 12.7 -- 0.22 -- -- TOCOPHEROL
B-SITOSTEROL mg/100 g 56.3 -- 6.51 26.4 3.81 BETA mg/100 g -- -- --
-- -- TOCOPHEROL BRASSICASTEROL mg/100 g 131 -- -- -- --
CAMPESTEROL mg/100 g 16.8 11.9 6.29 3.72 8.03 8.08 CHOLESTEROL
mg/100 g -- -- -- -- -- DELTA mg/100 g 5.47 0.76 0.28 1.48 -- 0.81
TOCOPHEROL ERGOSTEROL mg/100 g 130 59.2 174 54.8 174 92 GAMMA
mg/100 g 2.25 -- 0.28 0.83 0.57 0.12 TOCOPHEROL STIGMASTEROL mg/100
g 18.7 6.19 16.3 13.3 15.7 11.6 OTHER STEROLS mg/100 g 279 111 151
139 98.3 130 ALPHA mg/g 0.11 0.18 0.17 TOCOTRIENOL BETA mg/g 0.02
0.04 <0.01 TOCOTRIENOL DELTA mg/g 0.06 <0.01 <0.01
TOCOTRIENOL GAMMA mg/g 0.02 0.03 0.07 TOCOTRIENOL TOTAL mg/g 0.21
0.25 0.24 TOCOTRIENOLS
Example 2
[0183] In the following examples and tables, algal biomass was
prepared from heterotrophically grown microalgae as described above
and in WO2008/151149, WO2010/063031, WO2010/045368, WO2010/063032,
WO2011/150411, WO2013/158938, 61/923,327 filed Jan. 3, 2014,
PCT/US2014/037898 filed May 13, 2014, and in U.S. Pat. No.
8,557,249. Biomass samples IIA to IIE of Table II were isolated
from various strains originating from Prototheca moriformis (UTEX
1435) that were prepared and cultured to achieve the indicated
fatty acid profile. Delipidated algal meal was prepared from dried
microalgal biomass as described above. Particle size was evaluated
with a Microtrac laser diffraction particle size analyzer.
TABLE-US-00002 TABLE II Biomass properties Biomass Sample
Delipidated algal meal IIA IIB (very IIC IID (very IIE (high IIF
(high (laurate) high oleic) (mid oleic) high oleic) oleic) oleic)
Assay Units S8162 S6697 S3150 S6697 S5587 S5587 C8:0 % 0.22 0.01
0.02 0.01 0.00 0.00 C10:0 % 17.18 0.11 0.02 0.11 0.01 0.01 C12:0 %
45.03 0.25 0.07 0.25 0.03 0.03 C14:0 % 11.16 0.52 1.95 0.52 0.41
0.41 C16:0 % 6.21 3.96 29.26 3.96 3.31 3.31 C18:0 % 1.12 2.85 2.77
2.85 2.22 2.22 C18:1 % 13.36 89.50 57.01 89.50 86.17 86.17 C18:2 %
4.72 1.16 6.66 1.16 5.50 5.50 C18:3 ALPHA % 0.46 0.20 0.33 0.20
0.24 0.24 Total Lipid by Weight % 62.2 58.45 56.3 18.93 11.85 9.17
Ash, AOAC 942.05 % 5.91 7.07 2.63 4.67 5.52 6.93 Protein, AOAC
990.03 % 3.37 3.08 2.36 4.62 6.27 5.41 Moisture, AOAC % 3.65 4.76
2.37 1.73 1.82 2.45 930.15 Fiber, AOAC 978.10 % 10.09 9.00 7.64
2.92 5.26 3.07 pH, AOAC 973.41 5.11 5.76 4.54 4.50 4.22 4.67 PS D10
micron 6.2 5.04 4.7 72 PS D50 micron 20.6 9.51 7.2 402 PS D90
micron 88.3 57.6 11.4 982
Example 3: Dispersions of Predried Algal Biomass in Water
[0184] This example describes a procedure used to achieve a
dispersion of a previously dried microalgal biomass in water that
is similar to that of undried cells. Particle size was evaluated
with a Microtrac laser diffraction particle size analyzer.
[0185] Upon growth in fermentation, cells of Prototheca moriforimis
UTEX 1435 were characterized by a particle size distribution shown
in Table III. Dried cells Prototheca moriformis formed 40-4,000 um
sized clusters in the form of a powdery flake. Dried microalgal
biomass was added to water at a loading of 15% by weight. The
mixture was then mixed with a low shear overhead mixer for 15
seconds. A uniform dispersion was obtained. The resulting solution
was then mixed with a Silverson stationary high shear mixer at
10,000 rpm for one minute. Table III shows wet particle size
distribution of the pre-dried microalgal biomass re-suspended in
water.
[0186] These results indicate that mixing techniques practiced were
sufficient to generate a particle size distribution that
approximates that of the pre-dried particle size distribution of
cells in fermentation broth.
TABLE-US-00003 TABLE III Particle size distribution Cells in
Fermentation Broth, Wet Suspension of Dried Particle Size Algae,
Wet Particle Size Percent Volume Cutoff (um) (um) d5 1.32 1.55 d10
1.60 1.92 d50 7.87 6.85 d90 11.33 13.45 d95 12.86 16.82
Example 4: Dry Films Prepared with Microalgal Biomass
[0187] This example describes formulations of microalgal biomass
lubricants and their coating onto heated aluminum to form
films.
[0188] Prior to formulation, dried microalgal biomass samples were
characterized by properties listed in Table II. Base lubricant
formulations were prepared according to recipes listed in Table IV.
Formulation components included carboxymethyl cellulose (FinnFix
LC) and surfactants such as Sodium Lauryl Sulfate (Ambion),
Tergitol Minfoam 1.times. (Sigma), and Tween20. A biocide, WT-22
(Anchor Drilling Fluids), containing formaldehyde and Proxel GXL
containing 1,2-benzisothiazolin-3-one in dipropylene glycol (Excel
Industries) were also examined. Proxel GXL was used at 10%-100% the
dosing amount of WT-22. When Proxel GXL was used instead of WT-22,
the weight percent of deionized water was adjusted accordingly (see
Table IV) to produce a lubricant formulation that totaled 100%.
WT-22 or Proxel GXL were both effective as biocides. Mixing of the
concentrated formulations was achieved with a Silverson overhead
high shear mixer. Upon mixing, the pH of each formulations was
raised to approximately 8.8-9.2 by addition of base (typically
NaOH, KOH, NH.sub.4OH, TEA or the like). Formulations were stored
in glass jars under ambient conditions until evaluated. These
formulae involved a 25% suspension of microalgal biomass, such that
a 9:1 dilution (10.times. dilution) with water would yield a 2.5%
microalgal biomass solution. The average particle size
distributions for 2.5% suspension of microalgal biomass (mid oleic
biomass of Table II) in water is shown in Table IVa.
TABLE-US-00004 TABLE IV Lubricant formulations prepared with
microalgal biomass Sample Component B-1 B-2 B-3 B-4 B-5 B-6 B-7 B-8
Dried microalgal biomass Weight % 25 25 25 25 25 25 25 25
Carboxymethyl cellulose (CMC) Component of 3 1 1 3 1 3 1 3 Tergitol
Minfoam 1X Formulation 0 0 0.5 0.5 0 0 0 0 Sodium Laureth Sulfate 0
0 0 0 0.5 0.5 0 0 Tween 20 0 0 0 0 0 0 0.5 0.5 WT-22 0 0 0.1 0.1
0.1 0.1 0.1 0.1 DI Water 72 76 73.4 70.4 73.4 70.4 73.4 70.4
TABLE-US-00005 TABLE IVa Particle size for aqueous lubricant
formulations with 2.5% intact microalgal biomass Distribution
Particle size (microns) D10 1.22 D50 7.19 D90 26.3
[0189] Upon mixing, each formulation displayed uniform suspension
over a two-day period. It was found that a CMC concentration
between 1% and 3% yielded a solution able to hold dried microalgal
cells in suspension. A Tergitol Minfoam 1.times. concentration of
0.5% yielded a surface tension suitable for coating metal and
mitigating the Leidenfrost effect.
[0190] Prior to spray coating evaluation, formulations were diluted
into water at a 9:1 dilution. The concentrated formulation was
weighed into a 50 mL conical. DI water was then added and the
mixture was shaken until uniform. With the aid of an external mix,
two fluid nozzle, diluted formulations were spray applied onto an
aluminum or steel platen heated to either 220.degree. C. or
320.degree. C. Each solution was atomized with an airline
pressurized to 18 psi. A spray angle of 45.degree. and a distance
of nine inches from the platen were selected for optimal coating.
An application rate of roughly 30 mL/min was used for 20 seconds to
deliver the microalgal formulation onto the platen.
[0191] Dried films were evaluated by light microscopy. Films formed
on an aluminum platen heated to 220.degree. C. were characterized
by largely intact encapsulated oil bodies with few free oil
droplets. Films formed on an aluminum or steel platen heated to
320.degree. C. in contrast were characterized by fewer intact
encapsulated oil bodies and far greater number of free oil
droplets. Both temperature regimes resulted in films that were dry,
stable, and resistant to physical disruption.
[0192] These results demonstrate conditions and formulations
comprising microencapsulated algal oil capable of generating solid
films adherent to a metal surface.
Example 5: Coefficient of Friction of Microalgal Biomass Under
Various Conditions as Determined by Steel Falex Pin and Vee Block
Tests
[0193] This example compares the lubricating properties of
formulations comprising microalgal biomass to those of formulations
with graphite under stresses relevant to metalworking fluids.
[0194] Microalgal biomass samples IIA, IIB, and IIC of Example 2,
heat treated biomass samples, as well as evaporated fermentation
broth were used in the formulations and testing described below.
Formulations were prepared according to recipes listed in Table IV.
Mixing of the concentrated formulations was achieved with a
Silverson overhead high shear mixer. pH was adjusted to
approximately 8.8-9.2 with concentrated NaOH. Formulations were
held in glass jars under ambient conditions until evaluated. Prior
to pin and vee evaluation, these formula were subsequently diluted
with water or used without dilution with water to the final solids
value listed in Table VI.
[0195] Different lubricant exposure methods were evaluated for
delivering formulations to the pin and vee apparatus. As noted in
Table VI, vee blocks were either immersed in the test lubricant
(wet), or were spray coated (dry) while being heated to different
temperatures using the procedure described in Example 4. Vee blocks
were coated while held under ambient conditions, or where noted,
while blocks rested on a hot plate heated to either 220.degree. C.
of 320.degree. C.
TABLE-US-00006 TABLE V Concentrated formulations Sample Component
D1 D2 D3 H1 H2 H3 H4 H5 H6 D4 D5 D6 D7 D8 Dried biomass Weight % 25
0 0 0 0 0 0 0 0 0 0 0 0 0 Sample IIB - Component S6697 Dried
biomass 0 25 0 0 0 0 0 0 0 0 0 0 0 0 Sample IIA - S8162 Dried
biomass 0 0 25 0 0 0 0 0 0 25 0 0 0 0 Sample IIC - S3150 Dried
biomass 0 0 0 25 0 0 0 0 0 0 25 0 0 0 Sample IIA - S8162 heated to
175 C. for 2 hrs Dried biomass 0 0 0 0 25 0 0 0 0 0 0 25 0 0 Sample
IIA - S8162 heated to 315 C. for 2 hrs Dried biomass 0 0 0 0 0 25 0
0 0 0 0 0 0 0 Sample IIC - S3150 heated to 175 C. for 2 hrs Dried
biomass 0 0 0 0 0 0 25 0 0 0 0 0 0 0 Sample IIA - S3150 heated to
315 C. for 2 hrs Dried biomass 0 0 0 0 0 0 0 25 0 0 0 0 0 0 Sample
IIB - S6697 heated to 175 C. for 2 hrs Dried biomass 0 0 0 0 0 0 0
0 25 0 0 0 0 0 Sample IIB - S6697 heated to 315 C. for 2 hrs
Evaporated 0 0 0 0 0 0 0 0 0 0 0 0 54.5 0 microalgal fermentation
broth, S3150 Carboxy methyl 0 0 0 0 0 0 0 0 0 1 1 1 1 0.5 cellulose
Tergitol 0 0 0 0 0 0 0 0 0 0.5 0.5 0.5 0.5 0.5 Minfoam 1X WT-22 0 0
0 0 0 0 0 0 0 0.1 0.1 0.1 0.1 0 Graphite 0 0 0 0 0 0 0 0 0 0 0 0 0
25 DI Water 75 75 75 73.4 75 75 75 75 75 73.4 43.9 73.4 43.9 74
TABLE-US-00007 TABLE VI Formulations evaluated by Pin (#8 Test Pin,
SAE3135 steel) and Vee Block (Standard Vee Block, AISI 1137 Steel)
Apparatus testing Final Test Vee Dry Film Formu- Percent Block
coating CoF Pin Run lation Solids exposure Temperature min Fail #
Sample Tested Type Exposure (.degree. C.) Plateau (lbs) 1 D1 25 Wet
0.074 N/A 2 D1 10 Wet 0.077 N/A 3 D1 5 Wet 0.069 N/A 4 D1 2.5 Wet
0.057 N/A 5 D2 25 Wet 0.063 N/A 6 D2 10 Wet 0.089 N/A 7 D2 5 Wet
0.074 N/A 8 D2 2.5 Wet 0.069 N/A 9 D2 2.5 Wet 0.069 N/A 10 H1 2.5
Wet 0.064 N/A 11 H3 2.5 Wet 0.061 N/A 12 D1 2.5 Wet 0.085 N/A 13 H5
2.5 Wet 0.070 N/A 14 H6 2.5 Wet 0.087 926 15 D3 2.5 Wet 0.081 N/A
16 H3 2.5 Wet 0.051 N/A 17 H4 2.5 Wet 0.075 1575 18 D8 2.5 Wet
0.075 N/A 20 D8 2.5 Dry 0.741 151 21 D2 2.5 Dry 0.108 514 22 D1 2.5
Dry 0.068 698 23 D3 2.5 Dry 0.064 568 24 D8 2.5 Dry 0.141 428 26 D4
2.5 Dry 0.108 385 27 D4 2.5 Dry 0.107 329 28 D4 2.5 Dry 220 0.063
728 29 D4 2.5 Dry 220 0.063 707 30 D4 2.5 Dry 320 0.082 536 31 D4
2.5 Dry 320 0.072 605 32 D8 10 Dry 0.240 258 33 D8 10 Dry 0.200 343
34 D8 10 Dry 220 0.134 592 35 D8 10 Dry 220 0.120 657 36 D8 10 Dry
320 0.112 734 37 D8 10 Dry 320 0.114 840 38 D7 2.5 Dry 220 0.075
581 39 D7 2.5 Dry 220 0.067 514 40 D7 2.5 Dry 320 0.096 694 41 D7
2.5 Dry 320 0.107 681 N/A indicates that pin failure was not
reached and that the test ran to the 3000 lbf limit of the
machine.
[0196] Runs 1-18 were conducted such that liquid lubricant samples
were exposed to the Falex pin and vee apparatus by full submersion.
For runs 1-8, formulations were prepared with dried microalgal
cells from either a high oleic content producing strain or a high
lauric content producing strain. These formulations were
characterized by coefficients of friction less than 0.08. Run 18
evaluated aformulation comprising graphite. In the full submersion
Falex test, this formulation was characterized by a coefficient of
friction of 0.075.
[0197] Runs 9-17 interrogated formulations prepared with dried
microalgae that were heated to temperatures 175.degree. C. or
315.degree. C. for two hours prior to formulation. The heat exposed
biomass was then suspended in water to a final concentration of
2.5% by weight. The resulting solutions were tested via the
submerged pin and vee assay.
[0198] Runs 20-41 evaluated dry film coatings applied to vee
blocks. Application was conducted either under ambient temperature,
or while the vee blocks were heated to the temperatures indicated.
The results show that the algal biomass film formulations achieve a
lower coefficient of friction than the graphite film across all
temperatures evaluated. As compared to graphite, the microalgal
biomass samples show increased pin stability at ambient and
220.degree. C. exposure, but decreased pin stability at 320.degree.
C.
Example 6: Dried Algal Biomass Demonstrates Low Volatile Organic
Compounds
[0199] Dry encapsulated oil powder was subjected to test method
ASTM E1868-10, Standard Test Method for Loss-On-Drying by
Thermogravimetry. This test method was developed for metalworking
fluids and direct-contact lubricants. Two different preparations of
dried microalgal encapsulated oil were characterized by VOCs of
7.88 g/L (0.788%) and 9.16 g/L (0.916%).
Example 7: Floor Sweep Composition Comparison Test
[0200] A comparison test was developed to evaluate the performance
of various floor sweep compositions against different dust and
liquid targets. The testing apparatus consisted of five parallel
lanes, each lane bounded by two 6 foot long solid metal strips. The
strips were affixed to floor surface at intervals approximately 5.5
inches wide. Each lane was measured into five zones in order, a
deposit zone, an advancing zone, a pick-up zone, a push thru zone,
and a final evaluation zone.
[0201] At the beginning of each test, equivalent mass samples of
various floor sweep compositions were deposited in the deposit
zone. Equivalent masses of `substrate` dust or liquid samples were
deposited along each lane in the pick-up zone. The test substrate
was applied was 1/3 the mass amount of floor sweep formulation
tested.
[0202] With a 30-inch wide nylon boom, three brush strokes were
exerted to advance the floor sweep compositions along the test
zones of the floor surface. The brush first stroke moved floor
sweep compositions from the deposit zone through the advancing
zone. The second moved floor sweep compositions from the advancing
zone through the pick-up zone. The third brush stoke moved the
floor sweep compositions from the end of pick-up zone through to
the final evaluation zone. Photographs of the test in progress were
collected before test commencement, between brush strokes, and
after test conclusion. Qualitative evaluations were noted.
Example 8: Improved Floor Sweep Compositions with Microalgal
Biomass
[0203] This example describes the preparation of floor sweep
compositions comprising microalgal biomass and their evaluation
against conventional commercial floor sweep compositions.
[0204] Floor sweep compositions were prepared by combining the
ingredients listed in Table XVI according to the weight percentages
indicated. Ingredients were added to a heavy duty plastic bag then
hand blended for 2 minutes. Dried algal biomass Sample C and
delipidated algal meal Sample F of Example 2 were used in these
formulations and were characterized by the properties listed in
Table VII. Quikrete All Purpose Sand, corn cobs, hard wood saw
dust, and conventional mineral oil or soybean oil floor sweep
compositions were obtained commercially.
TABLE-US-00008 TABLE VII Floor sweep formulations Weight Sample %
of Formu- Formu- lation Descriptor Ingredients lation FS1 Biomass
& Sand Algal Biomass Sample 25 C - S3150 Quikrete All Purpose
75 Sand FS2 Blended Algal Biomass Sample 12.5 biomass & sand C
- S3150 Delipidated Biomass 12.5 Sample F Quikrete All Purpose 75
Sand FS3 Delipidated Delipidated Biomass 25 biomass & sand
Sample F Quikrete All Purpose 75 Sand FS4 MMC Green Sawdust 60 Sand
20 Soybean oil 20 FS5 MMC Mineral Oil Sawdust 60 Sand 20 Mineral
oil 20 FS6 Blended Algal Biomass Sample 12.5 biomass & C -
S3150 saw dust Delipidated Biomass 12.5 Sample F Saw dust 75 FS7
Blended Algal Biomass Sample 12.5 biomass & C - S3150 corn cobs
Delipidated Biomass 12.5 Sample F Corn cobs 75 FS8 Delipidated
Delipidated Biomass 25 biomass & Sample F corn cobs Corn cobs
75 FS9 Blended Algal Biomass Sample 12.5 biomass, C - S3150 corn
cobs Delipidated Biomass 12.5 and sand Sample F Corn cobs 60
Quikrete All Purpose 15 Sand
[0205] The floor sweep formulations of Table VII were evaluated by
the test methodology outlined in Example 7. In this example, tracks
of the testing apparatus were affixed to an unpolished concrete
floor. Substrates challenged by the formulations are listed in
Table VII along with a score that reflects formulation ease of
advancement along the floor surface as well as absorbance of the
target substrate. Scores are relative to a commercial, mineral oil
based floor sweep composition. A score above 1 indicates improved
performance, a score below indicates disadvantaged performance, and
a score equal to 1 indicates equivalent performance relative to the
commercial mineral oil based standard. Sets of samples and targets
that were not assessed are indicated in Table VII as `n.a.`.
TABLE-US-00009 TABLE VIII Qualitative ranking results of floor
sweep formulation testing Floor Sweep Substrate Sample Wheat Wood
Used Formulation Descriptor Flour Flour Talc Water Motor Oil FS4
MMC Green 0 1 0 0 -1 FS5 MMC Mineral Oil 1 1 1 1 1 FS1 Biomass
& Sand 1 n.a. n.a. n.a. n.a. FS2 Blended biomass & sand 0 2
1 0 2 FS3 Delipidated biomass & sand -1 n.a. n.a. n.a. n.a. FS6
Blended biomass & sawdust n.a. 2 1 3 3 FS7 Blended biomass
& corn cobs n.a. 2 1 3 3 FS8 Delipidated biomass & corn
cobs n.a. 1 1 2 2 FS9 Blended biomass, corn cobs and sand n.a. 1 1
0 2
[0206] The results presented in Table VIII demonstrate that various
floor sweep compositions comprising algal biomass tested against
different floor sweep substrates show improved surface floor
advancement and improved absorbance conjunction with different test
substrates relative to conventional, commercial floor sweep
formulations. Compositions with algal biomass are equivalent or
more effective than conventional floor sweep formulations at
removing talc from concrete floor surfaces. Compositions with algal
biomass and either sawdust or corn cobs but without sand are more
effective than conventional floor sweep formulations at removing
water from concrete floor surfaces. Compositions with algal biomass
and combinations of saw dust, corn cobs, or sand are more effective
than conventional floor sweep formulations at removing used motor
oil from concrete floor surfaces.
Example 9: Improved Absorbance Capacity of Floor Sweep Compositions
with Microalgal Biomass
[0207] This example compares the water and oil absorbance
properties of microalgal biomass and floor sweep compositions
comprising microalgal biomass to those of conventional floor sweep
ingredients and conventional floor sweep compositions.
[0208] Floor sweep ingredients as well as blended floor sweep
compositions were obtained or generated according to the procedures
indicated Example 8. Five grams of each ingredient or formulation
listed in Table IX was weighed into sets of paired 50 ml conical
centrifuge tubes. 30 mls of room temperature H.sub.2O was added to
one set of tubes, 20 mls of room temperature vacuum pump mineral
oil was added to the second set of tubes. The suspensions were
mixed by vortex mixer for 2 minutes then allowed to rest at ambient
temperature for 1 hour. Suspensions were then centrifuged for 10
minutes at 12,000 g. Unabsorbed liquid from each sample was
decanted. Pellets were then weighed. Fold absorbance was measured
and is represented by the following formulation: ([(Mass of pellet
after test)-(initial mass of sample evaluated)]/(initial mass of
sample evaluated)).
TABLE-US-00010 TABLE IX Water and Oil Absorbance of Floor Sweep
Ingredients and Compositions Weight % in Fold Formulation Formu-
Absorbance Sample Descriptor Ingredients lation Water Oil AS1
Blended Algal Biomass Sample 12.5 2.27 0.76 biomass, C - 3150 corn
cobs Deplidated Biomass 12.5 and sand Sample F Kwikrete
multipurpose 37.5 sand Cornsorb Corncobs 37.5 AS2 Blended Algal
Biomass Sample 12.5 2.42 0.89 biomass C - 3150 and corn Deplidated
Biomass 12.5 cobs Sample F Cornsorb Corncobs 75 AS3 Blended Algal
Biomass Sample 12.5 1.78 1.1 biomass, C - 3150 corn cobs Deplidated
Biomass 12.5 and sand Sample F Kwikrete multipurpose 15 sand
Cornsorb Corncobs 60 AS4 Blended Algal Biomass Sample 12.5 1.62
0.59 biomass, C - 3150 sawdust Deplidated Biomass 12.5 Sample F
Smith Company 75 Hammer milled sawdust AS5 Blended Algal Biomass
Sample 12.5 2.18 0.57 biomass, C - 3150 sawdust Deplidated Biomass
12.5 and sand Sample F Smith Company 60 Hammer milled sawdust
Kwikrete multipurpose 15 sand AS6 Blended Algal Biomass Sample 12.5
0.63 1.48 biomass C - 3150 and sand Deplidated Biomass 12.5 Sample
F Kwikrete multipurpose 75 sand AS7 Sand Kwikrete multipurpose 100
0.3 1.63 sand AS8 Sawdust Smith Company 100 2.7 0.25 Hammer milled
sawdust AS9 Corncobs Cornsorb Corncobs 100 3.03 0.99 AS10 Algal
Algal Biomass Sample 100 0 0.68 Biomass C - 3150 Sample C - 3150
AS11 Deplidated Deplidated Biomass 100 1.51 1.21 Biomass Sample F
Sample F AS12 Green Sawdust 59 0.6 1.37 Commercial Sand 20 Floor
Sweep Wax 20 polyacrylamide 1 superabsorbent AS13 Mineral oil
Sawdust 70 1.3 0.8 Commercial Mineral oil 30 Floor Sweep AS14
Mineral Oil Sand 20 0.82 1.35 Commercial Sawdust 60 Floor Sweep
Mineral oil 20
[0209] The results presented in Table IX demonstrate that various
floor sweep compositions comprising algal biomass show improved
water or oil fold absorbance relative to conventional, commercial
floor sweep formulations. Samples AS1-AS5, comprising a blend of
algal biomass, delipidated algal meal, and other ingredients were
characterized by an improved fold water absorbance (ranging from
1.62-2.42) relative to the fold absorbance of commercial floor
sweep compositions (0.6-0.8 fold). Sample AS6, a blend of algal
biomass, delipidated algal meal, and sand was characterized by an
equivalent or improved fold oil absorbance (1.48 fold) relative the
fold absorbance of commercial floor sweep compositions (0.8-1.37
fold).
Example 10: Reduced Friction and Wear with Algal Biomass
Formulations in Water
[0210] This example compares the friction reduction and wear
properties of formulations containing microalgal biomass to those
of formulations with graphite or molybdenum disulfide under
stresses relevant to metalworking fluids.
[0211] Prior to formulation, dried microalgal biomass samples were
characterized by properties listed in Table II. Powder forms of
solid lubricants were obtained from commercial sources: graphite
(Asbury Carbon) and molybdenum disulfide (Climax Molybdenum).
Powdered graphite was characterized by a particle size range of
0.5-50 micons. Powdered molybdenum disulfide was characterized by a
particle size range of 0.5-5 micons. Base lubricant formulations
were prepared according to recipes listed in Table X. Mixing of the
concentrated formulations was achieved with a Silverson overhead
high shear mixer or a low shear overhead mixer until the mixture
was uniform. The pH of each formulation was raised then to
approximately 8.8-9.2. Formulations were stored in glass jars under
ambient conditions until evaluated. These formulae involved a 25%
suspension, such that a 9 part water to 1 part formula dilution
yielded a 2.5% solids solution, thus generating samples G-1
(containing 2.5% microalgal biomass), G-2 (containing 2.5%
graphite), and G-3 (containing 2.5% MoS.sub.2). Diluted
formulations (2.5% solids) were evaluated according to ASTM D 3233
Method A, ASTM D 2670, ASTM D 4172, and ASTM D 2783. Results of
these standardized tests are listed in Table XI.
TABLE-US-00011 TABLE X Lubricant formulations Sample Component H-1
H-2 H-3 Dried microalgal biomass Weight % 25 0 0 Synthetic Dry
Graphite Component 0 25 0 Super Fine Molybdenum 0 0 25 Disulfide
Carboxymethyl cellulose 2 2 2 Proxel .TM. GTL (Lonza) 0.05 0.05
0.05 DI Water 72.95 72.95 72.95
[0212] Diluted formulations (2.5% solids) were evaluated according
to extreme pressure and wear tests ASTM D 3233 Method A, ASTM D
2670, ASTM D 4172, and ASTM D 2783. Results of these standardized
tests are listed in Table XI.
TABLE-US-00012 TABLE XI Results of Extreme Pressure and Wear
Standardized Tests Sample Test Measure G-1 G-2 G-3 ASTM D 2783,
Standard Weld point 126 126 400 Test Method for (kg) Measurement of
Extreme- Last Non 50 50 63 Pressure Properties of Seizure
Lubricating Fluids (Four- Load (kg) Ball Method) Wear Index 19.07
27.74 90.9 ASTM D 4172, Standard Average 1.046 1.682 1.254 Test
Method for Wear Scar Preventive Characteristics Diameter of
Lubricating Fluid (Four- (mm) Ball Method) ASTM D 2670, Standard
Tooth Wear 13 48 39 Test Method for Measuring (Teeth) Wear
Properties of Fluid Lubricants (Falex Pin and Vee Block Method)
ASTM D 3233 Method A, Coefficient 0.047 0.121 0.056 Standard Test
Methods for of Friction Measurement of Extreme (min) Pressure
Properties of Load at no 2536 no Fluid Lubricants (Falex Pin
Failure fail fail and Vee Block Methods) (lbs)
[0213] The results presented in Table XI demonstrate that the
formulation prepared with microalgal biomass was characterized by
reduced wear relative to those prepared with graphite or molybdenum
disulfide. The wear results of ASTM D 2670 demonstrate that the
formulation with microalgal biomass was characterized by two fold
or lower wear in relation to formulations with either graphite or
molybdenum disulfide. The wear results of ASTM D 4172 demonstrate
that the formulation with microalgal biomass was characterized by
37% wear reduction relative to the formulation with graphite and a
16% wear reduction relative to the formulation with molybdenum
disulfide.
[0214] The ASTM D 3233 Method A results presented in Table XI
demonstrate that the formulation prepared with microalgal biomass
was characterized a lower coefficient of friction relative to the
formulations prepared with graphite or with molybdenum
disulfide.
Example 11: Reduced Friction with Algal Biomass Formulations in
Oil
[0215] This example compares the friction reduction and extreme
pressure properties of oil-based formulations containing microalgal
biomass, microalgal oil, or microalgal delipidated meal under
stresses relevant to metalworking fluids.
[0216] Prior to formulation, dried microalgal biomass and
microalgal delipidated meal samples were characterized by
properties listed in Table II with the exception that both dried
biomass and delipidated biomass were prepared to a final average
particle size below 100 microns. Microalgal oil was characterized
by properties listed in Table I, Sample IF (S6697). Petroleum
derived Group II base oil, fumed silica, and bismuth octoate were
obtained from commercial sources. Weight based formulations were
prepared according to the recipes listed in Table XII. Mixing of
sample formulation was achieved with an overhead low shear mixer
utilizing a Cowles blade followed by an overhead high shear
Silverson mixer until the mixture was uniform. Formulations were
stored in glass jars under ambient conditions until they were
evaluated according to the extreme pressure test ASTM D 3233 Method
A, allowing the load to increase until pin failure. In the absence
of pin failure, a load of 3,000 lbs or more was applied. Results of
this standardized test are shown in Table XIII
TABLE-US-00013 TABLE XII Oil-Based Lubricant Formulations Sample
Component I-1 I-2 I-3 I-4 Group II Paraffinic Base Weight % 97.7
96.2 95.2 96.7 Oil Component Microalgal Oil (S6697) of 0 1.5 0 0
Dried microalgal biomass Formulation 0 0 2.5 0 Delipidated
microalgal 0 0 0 1 biomass Fumed Silica 0.1 0.1 0.1 0.1 Bismuth
Octoate 2.2 2.2 2.2 2.2
TABLE-US-00014 TABLE XIII Results of Extreme Pressure Standardized
Tests Sample Test Measure I-1 I-2 I-3 I-4 ASTM D 3233 Method A,
Load at Failure 202 520 no no Standard Test Methods for (lbs) fail
fail Measurement of Extreme Pressure Properties of Fluid Lubricants
(Falex Pin and Vee Block Methods)
[0217] The results presented in Table XIII demonstrate that the
formulations prepared with microalgal biomass or with delipidated
microalgal biomass in addition to fumed silica and bismuth octoate
were able to lubricate the spinning pin to be able to withstand a
load of 3,000 or greater. In contrast, formulations with microalgal
oil or with Group II base oil alone, in addition to fumed silica
and bismuth octoate, were unable to lubricate the pin above loads
of 520 lbs.
Example 12: Twist Compression Tests with Algal Biomass
Formulations
[0218] This example compares the friction reduction and load
properties of formulations containing microalgal biomass to those
containing graphite under stresses relevant to metalworking
fluids.
[0219] Prior to formulation, dried microalgal biomass samples were
characterized by properties listed in Table II. Powdered graphite
was obtained from Asbury Carbon. Lubricant formulations were
prepared according to recipes listed in Table XIV. Mixing of the
formulations was achieved with a low shear mixer followed by a
Silverson overhead high shear mixer until the mixture was uniform.
The pH of each formulation was raised then to approximately
8.8-9.2. Formulations were stored in glass jars under ambient
conditions until evaluated.
TABLE-US-00015 TABLE XIV Formulations Sample Component J-1 J-2
Dried microalgal biomass Weight % 25 0 Synthetic Dry Graphite
Component 0 25 Carboxymethyl cellulose of 2 2 Proxel .TM. GTL
(Lonza) Formulation 0.05 0.05 DI Water 72.95 72.95
[0220] The twist compression test was employed on dilutions of
samples listed in Table XIV to evaluate the coefficient of friction
of dry films adhered to aluminum 6061 and steel W-1 plates. Prior
to evaluation, samples J-1 and J-2 were diluted in 3 parts water to
1 part formulation (4.times. dilution) to obtain formulations K-1
(microalgal biomass) and K-2 (graphite) with 6.25% solids. Aluminum
6061 plates, heated to 100.degree. C., were spray coated with
either K-1 or K-2 formulations. Films were allowed to dry under
ambient conditions. An annular tool was then rotated at 10 rpm
under pressure over the aluminum 6061 or steel W-lplates on which
the test lubricants had been spray applied. The pressure applied
ranged from 1,000-5,000 psi. Data was collected electronically and
the coefficient of friction was calculated from the ratio of
transmitted torque to applied pressure. Results of these tests, run
at the pressures indicated, are shown in Table XV.
TABLE-US-00016 TABLE XV Twist Compression Test Results Sample K-1
Sample K-2 AL 1,000 AL 3,000 AL 5,000 Steel 20,000 AL 1,000 AL
3,000 AL 5,000 Steel 20,000 Test psi psi psi PSI psi psi psi PSI
Initial peak 0.085 0.043 0.026 0.014 0.246 0.198 0.164 0.072 Time
to 279.7 230.46 85.17 296.98 298.74 287.94 10.12 59.07 breakdown
(sec) Coefficient of 0.071 0.055 0.034 0.017 0.22 0.199 0.176 0.054
Friction Twist 3790 4381 2629 18109 1327 1448 58 1026 Compression
Test Friction Factor AL--aluminum
[0221] The results presented in Table XV demonstrate that the dry
films prepared with microalgal biomass were characterized by a
lower coefficient of friction than those prepared with graphite. At
5,000 psi, coefficient of friction of sample K-1 on aluminum was
80% lower than that of sample K-2 on aluminum (0.034 vs 0.176). The
initial peak is the coefficient of friction when the test reaches
full pressure. At 5,000K psi, the initial peak of the microalgal
film sample was 84% lower than that of the graphite film sample.
"Twist compression test friction factor" is an aggregate measure of
the various results obtained from the twist compression test.
Higher values of the twist compression test friction factor
indicate that the lubricant provides more lubricity. As can be seen
above, the twist compression test friction factor for the
formulation comprising biomass when applied to steel and subjected
to 20,000 psi is 18,109, where for the formulation containing
graphite the twist compression test friction factor is 1026. This
is a greater than 17-fold increase in the twist compression test
friction factor indicating that the formulation comprising biomass
is a significantly better lubricant than the control lubricant
formulated with graphite. Similarly, the time to breakdown for
formulations comprising biomass is significantly greater. The time
to breakdown for aluminum at 5,000 psi is 85.17 (biomass
formulation) versus 10.12 (graphite formulation), an 8.4 fold
increase. Collectively, these data demonstrate the ability of
formulations prepared with microalgal biomass to achieve lower
friction on aluminum and steel surfaces than those prepared with
graphite.
Example 13: Reduced Friction with Algal Biomass Formulations in
Oil
[0222] This example compares the friction reduction and extreme
pressure properties of oil-based formulations containing microalgal
biomass to those of formulations containing graphite or molybdenum
disulfide under stresses relevant to metalworking fluids.
[0223] Prior to formulation, dried microalgal biomass was
characterized by properties listed in Table II with the exception
that it was prepared to a final average particle size below 100
microns. Suspended forms of solid lubricants were obtained from
commercial sources: graphite (Graphkote 495, Asbury Carbon) and
molybdenum disulfide (SLA 1286, Henkel). Petroleum-derived Group II
base oil, fumed silica, and bismuth octoate were obtained from
commercial sources. Weight based formulations were prepared
according to the recipes listed in Table XVI. Mixing of sample
formulation was achieved with an overhead low shear mixer utilizing
a Cowles blade followed by an overhead high shear Silverson mixer
until the mixture was uniform. Each of the formulations were
characterized by 2.5% solids content. Formulations were stored in
glass jars under ambient conditions. They were evaluated according
to the extreme pressure test ASTM D 3233 Method A, allowing the
load to increase until pin failure. In the absence of pin failure,
a load of 3,000 lbs or more was applied. Results of this
standardized test are shown in Table XVII.
TABLE-US-00017 TABLE XVI Oil-Based Lubricant Formulations Sample
Component L-1 L-2 L-3 Group II Paraffinic Base Weight % 95.2 72.7
89.2 Oil Component Dried microalgal biomass of 2.5 0 0 naGraphite
(Graphkote) Formulation 0 25 0 molybdenum disulfide 0 0 8.5 (SLA
1286) Fumed Silica 0.1 0.1 0.1 Bismuth Octoate 2.2 2.2 2.2
TABLE-US-00018 TABLE XVII Results of Extreme Pressure Standardized
Test Sample Test Measure L-1 L-2 L-3 ASTM D 3233 Method A,
Coefficient of 0.099 0.313 0.051 Standard Test Methods for Friction
at end Measurement of Extreme of test or at break Pressure
Properties of Load at Failure no 1007 no Fluid Lubricants (Falex
Pin (lbs) fail fail and Vee Block Methods)
[0224] The results presented in Table XII demonstrate that the
formulations prepared with microalgal biomass, fumed silica and
bismuth octoate were able to lubricate the spinning pin to be able
to withstand a load of 3,000 or greater and were characterized by a
coefficient of friction at the end of the test of 0.099. In
contrast, formulations with graphite, fumed silica and bismuth
octoate were unable to lubricate the pin above loads of 1007 lbs
and were characterized by a coefficient of friction of 0.313.
Example 14: Metal Removal Fluids with Microalgal Oil
[0225] This example describes the load carrying and lubricating
properties of chlorinated paraffin-free formulations comprising
microalgal oil under stresses relevant to metalworking fluids.
[0226] Prior to formulation, microalgal oil was characterized by
properties listed in Table I (Sample IF, S6697, >88% high oleic
content, <2% polyunsaturated content). Lubricant formulations
comprising extreme pressure, antioxidant, rust inhibitor, metal
deactivator, and viscosity modifier additives were mixed into a
vessel charged with microalgal oil to achieve an effective
viscosity. Two formulations, M-1 and M-2 were evaluated according
to ASTM D 3233 Method B. Results of these standardized tests are
listed in Table XVIII.
TABLE-US-00019 TABLE XVIII Results of Extreme Pressure Step Test
Formulation M-1 Formulation M-2 (82.7% Microalgal Oil (92%
Microalgal Oil S6697) S6697 and derivatives) Load Torque
Temperature Torque Temperature (lbs) (lbs force) (.degree. F.) (lbs
force) (.degree. F.) 300 7.85 85 6.8 87.5 500 9.25 86.5 8.9 98.5
750 12.55 91 10.8 103.5 1000 14.55 93.5 12.0 108.0 1250 16.3 100
13.2 113.5 1500 17.9 105.5 14.9 120.5 1750 19.6 111.5 15.5 127.5
2000 20.95 117 16.4 131.5 2250 21.8 124 17.5 137.5 2500 22.55 133
18.7 141.5 2750 23.5 138.5 19.9 148.0 3000 24.55 145 21.0 156.0
3250 24.85 152 22.9 163.5 3500 25.7 158.5 24.5 170.0 3750 26.1 165
25.7 176.0 4000 26.15 168.5 27.1 185.0 4250 27.15 172 27.4 194.0
4500 27.1 181
[0227] Results presented in Table XVIII demonstrate that
formulations with microalgal oil achieve loads of >4,000 lbs and
are free of chlorinated paraffins.
Example 15: Reduced Grease Additives with Microalgal Biomass
[0228] This example describes the load carrying and wear properties
of grease formulations comprising microalgal biomass.
[0229] Prior to formulation into greases, dried microalgal biomass
was characterized by properties listed in Table II. Weight based
grease formulations were prepared according to the recipes listed
in Table XIX. 12-hydroxy stearate lithium grease base, chlorinated
ester, and technical grade molybdenum disulfide were obtained from
commercial sources as indicated in Table XIX below. Grease
formulations were prepared by charging a Kitchen Aid Pro 600 with
pre-additized lithium 12 grease. The blender was brought to a
medium orbital speed of 40 rpm. The grease was then further charged
with either molybdenum disulfide chlorinated ester, sifting in to
assure dispersion. Mixing was allowed to proceed for 1 hour or
until a homogeneous grease blend was achieved. The grease
formulations as indicated were then further charged with dried
microalgal biomass. Mixing continued for a minimum of one hour.
Formulations were evaluated by cone penetration (ASTM D217) before
and after exposure to 1,000 cycles in a Koehler K18100 Grease
Worker. ASTM D 2266 Four-Ball wear testing was conducted on 20 gram
worked samples. Results of these standard tests are shown in Table
XX.
TABLE-US-00020 TABLE XIX Grease Formulations N-2 N-4 Grease Grease
N-1 with N-3 with Grease Chlorinated Grease Molybdenum with
Paraffin and with Disulfide and Chlorinated Microalgal Molybdenum
Microalgal Paraffin biomass Disulfide biomass Formulation wt % of
Formulation #2 Lithium 95 94.5 99 98.5 grease base (Battenfeld)
Chlorinated 5 3.5 0 0 Ester (Qualice) Molybdenum 0 0 1 0.5
disulfide (Gamay Ind.) technical 5 um X bar Dried 0 2 0 1
microalgal biomass
TABLE-US-00021 TABLE XX Results of ASTM D 2266: Wear Preventative
Pressure Characteristics of Lubricating Grease Grease Base with
Qualice Grease Base with Gamay Chlorinated Paraffin Ind. Molybdenum
Disulfide Measure N-1 N-2 N-3 N-4 Load wear 42 37 46 40 46 Index
Exteme 400 400 250 250 Pressure Weld (kg)
[0230] The results shown in Table XX demonstrate that microalgal
biomass may be used to lower the amount of chlorinated paraffin or
the amount of molybdenum disulfide in grease formulations while
maintaining near identical wear and weld properties.
Example 16: Reduced Wear with Microalgal Biomass
[0231] This example describes improved wear properties of
metalworking formulations comprising microalgal biomass.
[0232] Prior to formulation, dried microalgal biomass was
characterized by properties listed in Table II. Where indicated in
Table XXI, 10% by weight microalgal biomass was blended into 90% by
weight metalworking formulation. Formulations were blended with a
handheld Master Mix and then evaluated by ASTM D 2670, Standard
Test Method for Measuring Wear Properties of Fluid Lubricants
(Falex Pin and Vee Block Method). Tooth wear as well as final
torque and final temperature are provided in Table XXI.
TABLE-US-00022 TABLE XXI Metalworking Formulations and Results of
ASTM D 2670 Weight % Final Final Tooth Microalgal Torque
Temperature Wear ASTM D2670 Biomass (lb force) (.degree. F.)
(teeth) Battenfield Lithium 0 17.4 221 120 General Purpose Grease
10 15.4 222 22 Qualice Chlorinated 0 18.2 142 21 Tapping Fluid 10
17.9 149 6
[0233] The results shown in Table XXI demonstrate that microalgal
biomass may be used to reduce wear in grease and in tapping
fluids.
Example 17: Lubricant Formulations
[0234] Additional lubricant formulations are shown in Table XXII
below.
TABLE-US-00023 TABLE XXII Lubricant formulations Formulation
Components Water Based 25% microalgae; Concentrate 1.5% CMC
(FinnFix LC); 0.5% Tergitol min foam; 0.5% Proxel GXL; 72.5% Water;
NaOH to pH 9.5 Oil Based 25% microalgae; Concentrate 1% Hydrophilic
Fumed Silica (Cabosil M5); 74% Calsol 5550 (Calumet; Naphthenic
Oil, treated for color and volatiles) Water and Oil Based 25%
microalgae; Concentrate 12.5% Chemfac PB-184 (phosphate ester based
emulsifier); 12.5% deionized water; 1% Hydrophilic Fumed Silica
(Cabosil M5); 50% HC100 (Calumet Naphthenic Oil) Delipidated and
50% solids from pressing; acid/base digested 50% Water; microalgal
biomass H.sub.2SO.sub.4 as acid for digest; Concentrate NaOH as
base for digest
[0235] Although this invention has been described in connection
with specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
[0236] All references cited herein, including patents, patent
applications, and publications are hereby incorporated by reference
in their entireties, whether previously specifically incorporated
or not.
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