Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCING AND ALTERING MICROBIAL FERMENTATION PRODUCTS
USING NON-COMMONLY USED LIGNOCELLULOSIC HYDROLYSATES
This work was supported in part by USDA Award 2011-10006-30377 Integrated
Biorefinery at the Domtar Plymouth, North Carolina Pulp Mill, sub-award to
Kuehnle
AgroSystems.
FIELD OF THE INVENTION
The present invention relates to microbial fermentation methods for
synthesizing
useful products resulting from the incorporation of non-commonly used
lignocellulosic
derivatives into culture medium. In one embodiment, the present invention
relates to
fermentation methods employing heterotrophic and/or mixotrophic culturing of
microorganisms with softwood or hardwood lignocellulosic simplified sugar in
the presence
of a non-sugar agent that is a wood-derived lignocellulose hydrolysis process
or wood-
derived organic acid solution.
BACKGROUND OF THE INVENTION
Global impetus is growing to provide alternatives to using fossil fuels and
diminished
non-renewable resources as sources of products that are used in large
quantities. Such
products can be used for production agriculture and aquaculture, health care,
and industrial
applications, and can span from compounds or ingredients for feed, foods,
beverages, dietary
supplements, crop protection, and personal care to raw materials for chemical
manufacturing.
Non-limiting examples of components that comprise the products include
proteins, lipids,
pigments, industrial polymers and recombinant molecules. Manufacturing these
products
using suitable microorganisms, such as microalgae, to replace unsustainable or
problematic
products or ingredients currently used in the marketplace and to do so using
economic inputs
for production is valuable
The use of microalgae as biofactories to generate massive volumes of renewable
biomass and bioproducts at competitive prices requires availability of
abundant and relatively
inexpensive feedstocks for fermentative bioconversion (heterotrophic or
mixotrophic).
Aerobic fermentation by heterotrophic algae is performed using generally
similar fermentor
tanks and operations as seen for other microorganisms in industrial
fermentation facilities.
Fermentation is considered the most economical and scalable method of algae
production. In
such fermentation, light can be used for mixotrophic growth by facultative
heterotrophic
microalgae using fixed carbon as well photosynthesis as a carbon source.
Fermentation can
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also proceed in darkness using fixed carbon with no photosynthesis by
facultative or obligate
heterotrophic microalgae.
Plant-based cellulosic sugars are increasingly attractive sources for
feedstocks for use
in microbial fermentation. These are generally from agricultural wastes or
residues that
remain after harvest or processing, purposefully grown energy grasses or
invasive grasses,
and low cost forestry-based biomass. Some examples of agricultural wastes
include corn
stover, soybean stover, wheat straw, barley straw, rice straw, oat straw, oat
hulls, canola
straw, and sugar processing residues such as bagasse and beet pulp. Some
examples of
grasses include switch grass, sweet sorghum, Miscanthus, and cordgrass.
Forestry-based
biomass includes underutilized wood (hardwood and softwood) and forest
residues (bark,
etc.); purposefully grown energy feedstocks include certain short-rotation
hardwood coppice
crops, such as willow, poplar, robina, and eucalyptus.
Underutilized woody biomass can be obtained from the pulp and paper industry
that
processes wood for various uses, for example, printing and writing paper
grades, various
coated and uncoated specialties paper grades, tissue and toweling products,
paperboard,
medical packaging, absorbent and air laid non-woven products (such as diapers,
hygiene,
incontinence products), textile fibers, film, and sawn timber. These products
utilize many
types of wood that may comprise but are not limited to Northern Softwood (for
example
Lodgepole Pine, White/Engelmann Spruce, Jack Pine, Sitka Spruce, Norway
Spruce, and
Black Spruce); Northern Hardwood (for example Maple, Birch, Poplar); Southern
Softwood
(for example Loblolly Pine, Shortleaf Pine); Southern Hardwood (for example
Oak, Maple
and Poplar). The other wood-based biomass in the supply chain comprises but is
not limited
to debarking residues, chip screening residues, knots and pulp fibers. The
associated mills
can be of various types and can include chemical pulp mills (such as sulfate
mills and sulfite
mills) and chemical-mechanical pulp mills (such as TMP and CTMP mills).
Unlike agricultural wastes or energy grasses such as stover and switchgrass,
which
can be highly seasonal for a specific geography, and therefore, pose serious
logistical
challenges inherent in shifting among feedstocks for operation of a
biorefinery, large
quantities of wood-based biomass are available all year round at a given
location.
Advantageously, large quantities of wood that can be transformed into
cellulosic feedstock
can support on-site, year-round, low cost industrial scaling to generate
microbial biomass
required annually for high-volume applications. This in turn can displace
significant
quantities of petroleum-derived products and reduce reliance on
environmentally unsound
practices.
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Pulp and paper mills have access to an abundance of low cost wood. Reduction
to its
component wood sugars such as by hydrolytic treatment may make it a potential
feedstock
for production of valuable algal biomass as part of an integrated biorefinery.
Much higher
resistance to degradation of biomass from wood, compared to biomass from the
grasses and
agricultural wastes, means the upstream processing pre-treatments and
conditions as well as
the downstream saccharification processes and conditions are necessarily
different. While
the use of cellulosic sugars and cellulosic hydrolysates from some non-wood
sources is viable
for supporting microalgal biomass production, use of wood-derived
lignocellulosic
hydrolysates for algal production has not been achieved until the present
invention.
Wood cellulosic material and hemicellulosic material can be pre-treated and
hydrolyzed by several processes known in the art. Non-limiting examples for
producing
wood-based sugar can comprise a biomass pre-treatment, which mainly
fractionates biomass,
followed by hydrolysis in which some fractions of biomass are converted into
sugar. The
most common lignocellulosic biomass pre-treatment techniques include: (a)
physical (e.g.,
chipping, grinding, milling, etc.); (b) biological; (c) chemical (e.g., using
acids, alkalines,
solvents, ozone, peroxide, etc.); and (d) physico-chemical processes (e.g.,
steam explosion,
hot water extraction, ammonia fiber extraction, etc.). These processes yield
hydrolysates
comprised of monosaccharides- simplified hexose and pentose sugars such as of
glucan (C6),
xylan (C5), arabinan (C5), mannan (C6), and galactan (C6) - along with other
wood-derived
non-sugar constituents, co-products/by-products and process residuals that
carry over from
prior pre-treatment and treatment steps.
These other constituents, co-products/by-products, or residuals are considered
impurities and are recognized as inhibitors of microbial growth. One potential
shortcoming is
the resulting impurities and inhibitors in the wood hydrolysates that may be
incompatible
with cultivation of microbes, specifically, microalgae.
To overcome this shortcoming, sugars from lignocellulosic hydrolysates can be
further processed (i.e., detoxified or conditioned) into purified sugars to
yield
monosaccharide feedstocks devoid of the toxic impurities in unpurified
hydrolysate to
support microbial growth and bioconversion activities. For example, US patent
8,889,402
describes cultivating heterotrophically in the dark a genetically engineered
Chlorella
protothecoides on pure carbon feedstock. US patent 7,063,957 describes
cultivating
Chlorella zofingiensis grown on glucose and producing pigment. US 7,674,609
discloses
cultivating Crypthecodinium cohnii on reagent grade glucose and organic acid.
US patent
8,889,402 discloses that Scenedesmus armatus and Navicula pelliculosa grow
better when
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pure carbon sources are added sequentially than when added together at the
outset. Other
work describes sugar uptake inhibition or transporter repression in mixed
fixed carbon
feedstocks. However, utilization of reagent grade components cannot predict
performance of
unpurified components in a composite solution. Also, the nature of wood
lignocellulosic
hydrolysate is such that the various carbon sources are added together at the
outset as they are
not purified carbon streams.
Unpurified wood-derived lignocellulosic hydrolysates would be more convenient
feedstock for microbial conversions, to minimize equipment, time, and energy
inputs required
for the further fractionation, and purification steps into purified
components. While
researchers have suggested that cellulose hydrolysis solutions can be a low
cost substitute for
glucose as a carbon source in the fermentation process, they have also
recognized that wood
lignocellulosic hydrolysis is difficult and costly. Therefore, having
favorably altered profiles
of target products can increase the value of the algal product and therefore
enable greater
economic returns.
Forest based companies may also integrate options at mill sites to produce
organic
acids such as acetic acid from a partial stream of lignocellulosic hydrolysate
that can further
serve as preferred fermentation feedstock for certain microbes. A mill may
also choose to
condition a partial stream of hydrolysate that can serve as preferred
fermentation feedstock
for certain microbes, for example, with use of a metal salt as described in US
Patent
Application Publication No. 20110318798.
There are discriminating characteristics between hardwoods and softwoods, just
as
there are characteristic differences between wood and the herbaceous grasses
or sugar crop
processing residues. Not all plant cell walls have the same cellulose,
hemicellulose and
lignin contents and compositions. Consequently, these differences are expected
to give rise
to differences in lignocellulosic hydrolysates.
It is well known that the composition and structure of the softwood and
hardwood
hemicelluloses differ, with the major class of hardwood hemicelluloses being
the
glucuronoxylans. This xylan is 0-acetyl-(4-0-methylglucurono)-b-D-xylan, with
the xylan
backbone having glucuronic acid substituents. The content of glucuronoxylan in
hardwoods
is typically between 15 and 30% by weight. In some birches xylan content can
reach as high
as 35%. Also, unlike softwoods, partial acetylation may occur on the 2 or 3
positions of the
xylose backbone to yield, for example, seven acetyl residues per ten xylose
units. Xylosidic
bonds between xylose units are easily hydrolyzed by acids, in contrast to
linkages between
uronic acid groups and xylose that are very resistant. The acetyl groups are
easily cleaved by
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alkali. Hardwoods also usually contain small amounts (2-5%) of glucomannan. It
is
composed of f$-D-glucopyranose and P-D-mannopyranose linked by (1¨>4) bonds
and the
glucose to mannose residues are generally in the ratio of 1:2. Mannosidic
bonds between
mannose units are more rapidly hydrolyzed than the corresponding glucosidic
bonds and
glucomannan is easily depolymerized under acidic conditions.
The major class of softwood hemicelluloses is 0-acetyl-galactoglucomannan,
with the
glucose to mannose ratio of about 1:3, and the ratio of galactose to glucose
varying from 1:1
to 1:10. Softwood xylan is an arabino-(4-0-methylglucurono)xylan. In contrast
to
hardwood, the softwood xylan does not contain acetyl groups and is more highly
branched
and more acidic than the hardwood xylan. These side chains can be removed
under mild
acidic conditions in which the main xylose chain remains intact. The arabinose
and uronic
acid substituents stabilize the xylan chain against alkali-catalyzed
degradation. The lignin
fraction of softwoods such as pine is generally considerably more than in
temperate
hardwoods, although this is not always the case, and can be nearly double
compared to corn
stover. While most lignin can be filtered out, their presence in process
hydrolysates may
cause issues as seen in ethanol fermentations.
Preferably, a hydrolysate feedstock would be suitable for use by microalgae
that are
capable of complete utilization of the C5 and C6 sugars for maximum biomass
yield. Many
microalgae strains appear unable to utilize pentose and hexose during
fermentation. Some
species utilize xylose or other pentose sugars with increased productivity
only when grown in
the presence of light. Difficulty in a cell's utilization of the cellulose and
hemicellulose-
derived sugars has been addressed for some algae using genetic engineering,
for example, for
uptake or modification of polysaccharides as disclosed in US patent 8,889,402
and US patent
8,592,188; of cellodextrin as disclosed in US patent 8,431,360; or of pentose
as disclosed in
US patent 8,431,360 and US patent 8,846,352.
Growth of algae and production of algal products using wood-derived
lignocellulosic
hydrolysates has not been disclosed. Cellulosic feedstocks for algae have been
limited to
non-wood sources. US Patent Application Publication No. 20110306100 discloses
the use of
switchgrass and corn stover following fungal enzymatic digestion for obtaining
algal fatty
acids. US Patent Application Publication No. 20100151538A1 discloses the use
of
depolymerized cellulosic material selected from the group consisting of corn
stover,
Miscanthus, forage sorghum, sugar beet pulp, and sugar cane bagasse for
heterotrophic
cultivation of Prototheca, an obligate non-pigmented microalga. US Patent
Application
Publication No. 20090011480 and US patent 8,790,914 disclose use of
depolymerized
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cellulosic material selected from the group consisting of corn stover,
switchgrass, and sugar
beet pulp for heterotrophic cultivation of microalgae. Use of rice straw, for
mixotrophic
cultivation of Chlorella pyrenoidosa, and of wheat bran using the microalgae
Chlorella
vidgaris and Scenedesmus obliquus for mixotrophic or heterotrophic cultivation
have been
reported. However, the methods of producing microalgal biomass and products
using wood-
sourced lignocellulosic hydrolysates are not disclosed. United States Patent
Application
Publication No. 20092117569 discloses the use of source material that
originates from treated
wood pulp for cultivation of yeasts. Also, the use of yeast cannot substitute
for microalgae in
whole composition and in terms of the production of compounds, certain
compositions,
yields, or mixture of compounds required for target products, such as high
quality animal,
insect or fish feed, nutritional proteins, polysaccharides and lipids,
immunomodulatory
compounds, nutritional and fiber supplements, colorants, and recombinant
nucleic acids and
proteins.
BRIEF SUMMARY OF THE INVENTION
An embodiment of the invention provides a method of producing a culture medium
for culturing a microbe to produce a product of interest. The method of
producing a culture
medium comprises the steps of:
a) providing a lignocellulosic biomass, wherein the lignocellulosic biomass
comprises
a lignocellulosic compound,
b) hydrolyzing the lignocellulosic biomass to produce a lignocellulosic
hydrolysate,
wherein the lignocellulosic hydrolysate comprises a simplified sugar produced
from at least a
portion of the lignocellulosic compound,
c) optionally, separating the lignocellulosic hydrolysate into a first portion
and a
second portion and treating the second portion of the lignocellulosic
hydrolysate to convert a
portion of the lignocellulosic compound and/or the simplified sugar to a non-
sugar agent;
d) mixing the treated second portion of the lignocellulosic hydrolysate
comprising the
non-sugar agent with the first portion of the lignocellulosic hydrolysate,
e) producing a culture medium comprising the mixture obtained after step d).
In one embodiment, the non-sugar agent is an organic acid, an alcohol, a
micronutrient, a salt, a saponifiable or fatty acid compound, a furfural,
process water, a
protein, or any combination thereof
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The non-sugar agent can be an organic acid such as acetic acid, propionic
acid, citric
acid, fumaric acid, glycolic acid, lactic acid, malic acid, pyruvic acid,
succinic acid,
glucuronic acid, galacturonic acid, and ferulic acid
A further embodiment of the invention also provides a culture medium produced
according to the method described above, which is hereinafter referred to as
"a method of
producing a medium containing lignocellulosic hydrolysate."
A further embodiment of the invention provides a method for synthesizing a
product
of interest using fermentation. In one embodiment, the method comprises the
steps of:
a) providing a culture medium produced according to the method of producing a
medium containing lignocellulosic hydrolysate;
b) providing a microalgal cell that produces the product of interest;
c) culturing the microalgal cell in the culture medium to produce a microalgal
culture
from the microalgal cell; and
d) purifying the product of interest from the microalgal culture.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Overall production process for producing microalgal products using
wood-
derived lignocellulosic hydrolysates. Key components in the process are
highlighted. [10]
Providing a culture medium comprising wood-derived lignocellulosic hydrolysate
with a
simplified sugar in the presence of a non-sugar agent; [20] choosing to
convert (Y) some of
that hydrolysate into a wood-derived organic acid, to produce a feedstock
stream enriched for
a specific non-sugar agent, which can be optionally provided [30] into the
culture medium;
and/or choosing to not convert (N) some of that hydrolysate into an organic
acid; [40]
providing a microalgal cell, and optionally a second type of microbial cell,
to produce a
culture by a fermentation [50]. Algal cells can be selectively grown on
hydrolysate with
process residuals and/or on an enriched feedstock stream of wood-derived
organic acid to
generate product and even alter the product of interest [60], which is then
purified to produce
the desired microalgae-derived target products [70].
Figure 2. 0D750
profiles of KAS908 (Chlorella sorokiniana) grown
heterotrophically in three wood hydrolysates in replicated 96-well plates:
Southern Hardwood
Chips (SHC), Southern Pine Bleached Kraft (SPBK) and Southern Pine Finer chips
(SPFC).
Figure 3. Comparison of heterotrophic growth in replicated 50-mL flasks
measured
by 0D750 absorbance of KA5740 (Scenedesmus armatus) on Southern Pine Finer
Chips
(SPFC) hydrolysate and equivalent glucose concentration.
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Figure 4. Heterotrophic growth of KAS1101 (Rhodotorula glutinis ATCC 2527) in
replicated 96-well plates using different concentrations of Southern Hardwood
Chip (SHC)
hydrolysates with Yeast Extract-Peptone (YP) nutrients or YP medium with 20
g/L glucose.
Figure 5. Growth and sugar utilization (glucose and xylose uptake monitored by
HPLC) of KA5908 (Chlorella sorokiniana) under heterotrophic fermentation in a)
BSP
(Bleached Southern Pine) wood hydrolysates and b) C5 and C6 model sugars
standardized to
total sugars in BSP wood hydrolysates, performed in a 7-L batch fermentor.
Figure 6. Thin layer chromatographs for phospholipids, phosphatidylcholine
(PC)
and phosphatidylethanolamine (PE), from KAS908 (Chlorella sorokiniana) biomass
grown
heterotrophically in medium with varying glucose:nitrogen (w/w) ratios at day
3 of growth:
Columns 1-7 show extracts from biomass in dark shake flask using various
glucose:nitrogen
(w/w) ratios: 1=1:1, 2= 3:1; 3= 4:1; 4=5:1; 5 = 7:1; 6=9:1; 7=13:1; 8= KAS908
in F/2
(photosynthetic control under lights); Columns 9-12 show extracts from biomass
in dark 6-L
fermentor: 9= 4:1 glucose to nitrogen ratio; 10=model sugars 19.43 g/L glucose
and 8.06 g/L.,
xylose in 2F; 11= Bleached Southern Pine lignocellulosic hydrolysate; and 12=
heterotrophic
2F + 36 g/L glucose.
Figure 7. Glucose utilization of KAS908 (Chlorella sorokiniana) and KAS1101
(Rhodotorula glutinis) using Southern Hardwood Chips (SHC) lignocellulosic
hydrolysate
sugars.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the singular forms "a", "an" and "the" are intended to include
the
plural forms as well, unless the context clearly indicates otherwise.
Furthermore, to the
extent that the terms "including", "includes", "having", "has", "with", or
variants thereof are
used in either the detailed description and/or the claims, such terms are
intended to be
inclusive in a manner similar to the term "comprising". The transitional
terms/phrases (and
any grammatical variations thereof) "comprising", "comprises", "comprise",
"consisting
essentially of', "consists essentially of', "consisting" and "consists" can be
used
interchangeably.
The term "about" or "approximately" means within an acceptable error range for
the
particular value as determined by one of ordinary skill in the art, which will
depend in part on
how the value is measured or determined, i.e., the limitations of the
measurement system.
Where particular values are described in the application and claims, unless
otherwise stated
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the term "about" meaning within an acceptable error range for the particular
value should be
assumed. In the context of compositions containing amounts of ingredients
where the terms
"about" or "approximately" are used, these compositions contain the stated
amount of the
ingredient with a variation (error range) of 0-10% around the value (X 10%).
In the present disclosure, ranges are stated in shorthand, so as to avoid
having to set
out at length and describe each and every value within the range. Any
appropriate value
within the range can be selected, where appropriate, as the upper value, lower
value, or the
terminus of the range. For example, a range of 0.1-1.0 represents the terminal
values of 0.1
and 1.0, as well as the intermediate values of 0.2, 0.3, 04, 0.5, 0.6, 0.7,
0.8, 0.9, and all
inteiniediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-
1.0, etc. When
ranges are used herein, combinations and subcombinations of ranges (e.g.,
subranges within
the disclosed range), specific embodiments therein are intended to be
explicitly included.
The term "photoautotrophs" refers to an organism capable of synthesizing its
own
food from inorganic substances using light as an energy source.
Examples of
photoautotrophs include green plants and photosynthetic bacteria.
The term "facultative" refers to an organism that is capable of but not
restricted to a
particular mode of life. For example, a facultative anaerobe can synthesize
ATP by aerobic
respiration if oxygen is present, but is capable of fermentation or anaerobic
respiration if
oxygen is absent.
The term "facultative heterotroph" refers to a photoautotrophic organism that
is also
capable of utilizing organic compounds for growth and/or maintenance and/or
survival when
light energy is not sufficient or is absent. The term also encompasses
facultative heterotrophs
and descendants thereof that lose their capability to perform photosynthesis,
or acquire
defects that result in their inability to grow as phototrophs, or are enabled
to grow in the dark
through genetically engineering, including for trophic conversion or for
utilization of the
preferred carbon feedstock. The term "obligate heterotroph" refers to a cell
that is unable to
perform photosynthesis and requires an exogenous feedstock for survival.
Some
representative facultative heterotrophs and obligate heterotrophs that can
grow in the dark in
the presence of a carbon source are used in the method of the invention in a
non-limiting
manner. Examples of other microalgae are given below.
The term "axenic" refers to the state of a culture in which only a single
species,
variety, or strain of an organism is present and wherein the culture is free
of all other
organisms.
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The term "biomass" as used herein refers to a mass of living or non-living
biological
material and its derivatives and includes both natural and processed, as well
as natural
organic materials more broadly. Thus, "microalgal biomass," and "algal
biomass" refers to
material produced by growth and/or propagation of microalgal cells. "Woody
biomass" refers
to biomass from trees and shrubs. "Lignocellulosic biomass" refers to biomass
comprising
lignocellulose, for example, wood.
"Biomass production" or "biomass accumulation" means an increase in the total
number or weight of the cells of the organisms that are present in a culture
over time.
Biomass is typically comprised of cells; intracellular contents as well as
extracellular material
such as may be secreted or evolved by a cell; and can also be processed such
that a fraction of
the biomass is removed leaving residual biomass.
"Biorefinery" means a facility that integrates biomass conversion processes
and
equipment to produce fuels, power, and chemicals from biomass. A pulp and
paper mill
biorefinery uses woody biomass.
"Fed-batch fermentation" refers to a fermentation where one or more nutrients
are
supplied to the bioreactor during cultivation and in which the product remains
in the
bioreactor until the end of the fermentation run.
A "product of interest" is a substance synthesized by a cell. Examples of a
product of
interest include but are not limited to, proteins, lipids, carbohydrates,
biogases, volatile
materials, sugars, amino acids, isoprenoids, terpenes, or precursor thereof.
Such substances
may be synthesized constitutively by the organisms throughout growth and the
amount of the
substance in the culture may increase simply due to an increase in the number
of organisms.
Alternatively, the synthesis of such substances may be induced or altered in
response to
culture conditions or other environmental factors, for example, nitrogen
starvation or elevated
ammonium levels, or components from cellulosic hydrolysates.
"Protein" refers to full-length protein polymers or peptide fragments thereof.
As non-
limiting examples, protein as peptides can be antibiotics or promoters of gene
expression.
Protein can be used in whole biomass or delipidated microalgal meal for animal
and fish feed.
The product of interest can also be an amino acid. An amino acid can have
nutritional
value, for example, taurine.
The product of interest can also be a polysaccharide. A polysaccharide can
have
health value, for example as immunomodulatory, macrophage-stimulating or
humectant
properties such as beta-glucan or undefined exopolysaccharides.
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The amount of a product of interest accumulated over time relative to the
culture
volume and relative to their original amount is considered as "product
accumulation" that can
be measured or quantified such as by specific productivity or on a relative
basis compared to
a control culture.
The term "conditions favorable to cell division" or "conditions favorable to
vegetative
growth" mean conditions in which cells divide at a pace such that an
industrial production
run is completed in about 60 to 168 to 240 hours, preferentially in less than
240, 144, 120 or
96 hours, including a lag time of less than about 24 hours.
The term "co-culture", and variants thereof such as "co-cultivate," refer to
the
presence of two or more types of cells in the same fermentor or bioreactor.
The two or more
types of cells may both be microorganisms, such as microalgae, or may be a
microalgal cell
cultured with a different cell type. The culture conditions may be those that
promote growth
and/or propagation of the two or more cell types or those that facilitate
growth and/or
proliferation of one, or a subset, of the two or more cells types while
maintaining cellular
growth for the remainder.
The term "cultivated" or "cultivation" or "culturing" refers to the purposeful
fostering
of growth (increases in cell size, cellular contents, and/or cellular
activity) and/or propagation
(increases in cell numbers via mitosis) of one or more microbial or microalgal
cells by use of
intended culture conditions. The combination of both growth and propagation
may be termed
proliferation. Examples of intended conditions include the use of a defined
medium (with
known characteristics such as pH, ionic strength, and carbon source),
specified temperature,
oxygen tension, and growth in a fermentor or bioreactor. The term does not
refer to the
growth of microorganisms in nature or otherwise without intentional
introduction or human
intervention, such as natural growth of an organism.
The term "fermentor" or "bioreactor" or "fermentation vessel" or "fermentation
tank"
means an enclosed vessel or partially enclosed vessel in which cells are
cultivated or
cultured, optionally in liquid suspension. A fermentor or bioreactor of the
disclosure includes
non-limiting embodiments such as an enclosure or partial enclosure that
permits cultured
cells to be exposed to light or which allows the cells to be cultured without
the exposure to
light. The term "port", in the context of a vessel that is a fermentor or
bioreactor, refers to an
opening in the vessel that allows influx or efflux of materials such as gases,
liquids, and cells.
Ports are usually connected to tubing leading from the fermentor or
bioreactor.
The term "fermenter" refers to an organism that causes fermentation.
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The term "fixed carbon source" means a compound containing carbon that can be
used as a source of carbon and/or energy by an organism. Typically, a fixed
carbon source
exists at ambient temperature and pressure in solid or liquid form.
The term "organic acid" refers to one or more molecules that are organic
compounds
with acidic properties. The
most common organic acids are the carboxylic acids.
A "carboxylic acid" contains a carboxyl group distinct from sugar
carbohydrates such as
glucose commonly used in algal fermentation. Acetic acid is a two-carbon
carboxylic acid,
CH3COOH, commonly used in chemical manufacturing. As a chemical reagent,
acetic acid is
manufactured from petrochemical feedstock. Propionic acid (propanoic acid) is
a carboxylic
acid with the chemical formula CH3CH2COOH. The anion CH3CH2C00¨ as well as
the salts and esters of propionic acid are known as propionates (or
propanoates). Other such
acids can include but are not limited to citric, fumaric, glycolic, lactic,
malic, pyruvic, and
succinic acids.
"Sugar acids" and "chlorogenic acids" are also organic acids and can include
but are
not limited to glucuronic, galacturonic and other uronic acids, and ferulic,
with a carboxylic
acid functional group such as obtained in lignocellulosic derivatives. Organic
acids can be
used alone or in combination, such as in combinations that may occur naturally
in
lignocellulosic derivatives.
Bio-based organic acids can be sourced from microbial anaerobic or partial
anaerobic
digestion or fermentation processes as is known in the art.
The terms "heterotrophic conditions" and "heterotrophic fermentation" and
"dark
heterotrophic cultivation" or "dark heterotrophic culture" refer to the
presence of at least one
fixed carbon source and the absence of light during fermentation.
"Mixotrophic
fermentation" refers to cultivation in the presence of at least one fixed
carbon source and the
presence of light during fermentation.
"Lignocellulosic hydrolysis" or "saccharification" refers to a process of
converting
cellulosic or lignocellulosic biomass into monomeric sugars or
monosaccharides, such as the
hexose, glucose, and the pentose, xylose. "Saccharified" or "simplified" or
"depolymerized"
cellulosic or lignocellulosic material or biomass refers to cellulosic or
lignocellulosic material
or biomass that has been converted into monomeric sugars through
saccharification.
Saccharification also produces oligosaccharides that are oligomeric, short-
chain polymers of
monomeric sugars. Some sugars are C12 dimers composed of two C6 sugars. These
dimers
can also be a starting point for an engineered or for a natural algae or other
microbe and/or
for an algal/microbial combination.
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Solid state fermentation of woody biomass by fungi or polycultures is one
process
known in the art to produce hydrolytic enzymes which subsequently produce
sugar-rich and
even nitrogen-rich streams, either in phased steps or in simultaneous
saccharification as
feedstock for algal heterotrophic or mixotrophic culture.
"Model sugar" or "purified sugar" refers to monomeric or oligomeric sugars
that are
individual sugars, separate from other sugars, in a pure or reagent grade
compound.
"Lignocellulosic hydrolysate" or "cellulosic hydrolysate" refers to the
products of
saccharification and the process residuals.
"Process residuals" or "process impurities" and "process inhibitors refers to
non-
monosaccharide and non-oligosaccharide residuals from the wood lignocellulosic
hydrolysis
process, comprising but not limited to compounds selected from organic acids
(e.g., acetic,
formic, levulinic), aldehydes (e.g., furfural, 5-hydroxymethylfurfural,
vanillin), lignins, lignin
byproducts or derivatives, inorganic salts (e.g., sulfates, phosphates,
hydroxides), alcohols,
fatty acids, fatty alcohols, fats, waxes, polyesters (e.g., suberin),
terpenoids, alkanes, wood
extractives, Hibbert's ketones, and proteins; where the organic acids may
further
comprise citric, fumaric, glycolic, lactic, malic, proprionic, pyruvic,
succinic, glucuronic,
galacturonic, uronic, chlorogenic, or lignocellulosic acid; and where the
solvent water is also
considered a process residual.
The term "feedstock" refers to nutritional material assimilated or metabolized
by a
cell.
The term "isoprenoid" or "terpenoid" or "terpene" or "derivatives of
isoprenoids"
refers to any molecule derived from the isoprenoid pathway with any number of
5-carbon
isoprene units, including compounds that are monoterpenoids and their
derivatives, such as
carotenoids and xanthophylls. The isoprenoid pathway generates numerous
commercially
useful target compounds, with non-limiting examples such as pigments,
terpenes, vitamins,
fragrances, flavorings, solvents, steroids and hormones, lubricant additives,
and insecticides.
These in turn are used in products for food and beverages, perfumes, feed,
cosmetics, and raw
materials for chemicals, nutraceuti cal s, and pharmaceuticals.
The term "carotenoid" refers to a compound composed of a polyene backbone
which
condensed from five-carbon isoprene unit, "carotenoid" can be an acyclic, or
one
(monocyclic) or two and it can be terminated by cyclic end-groups of the
number (bicyclic).
The term "carotenoid" may include both carotenes and xanthophylls.
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A "carotene" refers to a hydrocarbon carotenoid. "Xanthophylls" are oxygenated
carotenoids. Modification of pyrophosphate and phosphate groups of isoprene
derivatives
include oxidations or cyclizations to yield acyclic, monocyclic and bicyclic
terpenes
including monoterpenes, diterpenes, tripterpenes, or sequiterpenes, etc.
"Lipids" refers to any of a large group of organic compounds that are oily to
the touch
and insoluble in water. Lipids include fatty acids, oils, waxes, sterols,
polar lipids, neutral
lipids, phospholipids, and triglycerides. They are a source of stored energy
and are a
component of cell membranes. Phospholipids are a lipid containing a phosphate
group in its
molecule. They include diacylglyceride structures, e.g., phosphatidic acid
(phosphatidate;
PA), phosphatidylethanolamine (cephalin; PE), phosphatidylcholine (lecithin;
PC),
phosphatidylserine (PS), phosphosphingolipids, and glycerophospholipids.
"PUFA" or
"PUFAs" refers to lipids that are polyunsaturated fatty acids. Examples of
PUFAs are
docosahexaenoic acid (D1-1A, represented as 22:6 n-3); eicosapentaenoic acid
(EPA,
represented as 20:5 n-3); omega-3 docosapentaenoic acid (DPA n-3, represented
as 22:5 n-3);
omega-6 arachidonic acid (ARA, represented as 20:4 n- 6); and omega-6
docosapentaenoic
acid (DPA n-6, represented as 22:5 n-6).
The term "microorganism" or "microbe" refers to microscopic unicellular
organisms,
including microalgae, which can also be filamentous or colonial. The
microorganisms usable
in the fermentation according to the present invention can include mutants,
naturally
occurring strains selected for a specific characteristic, or genetically
engineered variants of a
naturally occurring strain.
The term "microalgae" refers to a eukaryotic microorganism that contains a
chloroplast, and optionally is photosynthetic, or a prokaryotic microorganism
capable of
being photosynthetic. Microalgae include obligate photoautotrophs, which are
incapable of
metabolizing a fixed carbon source as energy, as well as obligate or
facultative heterotrophs,
which are capable of metabolizing a fixed carbon source. Microalgae as
obligate
heterotrophic microorganisms include those that have lost the ability of being
photosynthetic
and may or may not possess a chloroplast or chloroplast remnant. Microalgae
can divide to
produce populations of cells and can be scaled-up or enter a production phase
to produce
biomass, and this process can be continued indefinitely until a maximum
productivity is
achieved.
The term "recombinant" when used with reference, a cell, nucleic acid,
protein, or
vector, indicates that the cell, nucleic acid, protein or vector, has been
modified from its
natural state. For example, a recombinant cell comprises an exogenous nucleic
acid or
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protein or the alteration of a native nucleic acid or protein, or is derived
from a cell or
organism or micro-organism so modified.
The term "robust" or "robust culture", in the context of selected strains or
lines of a
species, refer to a population of algae that contain a desired phenotype and
equal or greater
growth characteristics, especially under heterotrophy, compared to the
original strain.
A method for use of wood-derived lignocellulosic hydrolysate directed towards
accumulation of sufficient biomass, target compound, or improved compound
profile by a
microalgae species will have economic benefits and for the first time,
demonstrate using a
non-seasonal agricultural resource that is available all year round for
efficient operations at a
mill-based biorefinery. In particular, methods to produce and modify levels of
target
compounds are desirable for optimizing efficient industrial heterotrophic
fermentation that is
independent of weather, climates, seasons, and geography. Target compounds of
value
include proteins, lipids, carotenoids/isoprenoids and recombinant molecules.
The latter may
be compounds which favor rapid biomass growth for their expression and
accumulation.
Surprisingly, it was discovered that the mixture of sugar and non-sugar agents
in the wood
lignocellulose hydrolysate favors production of algal product biomass over use
of pure sugars
alone; shortens fermentation cycle time, increases yield; alters protein yield
and composition;
alters lipid yield and composition; supports recombinant gene expression;
induces and
supports certain pigment accumulation in a dark fermentation; reduces certain
other pigment
accumulation in a dark fermentation; and enables co-culture of two different
species to fully
utilize the fixed carbon component.
In one embodiment, the invention provides that not only does wood-derived
lignocellulosic hydrolysate support algal growth, but some algal species also
perform better
in the presence of unpurified wood hydrolysate and the resulting products can
differ in
several ways. For example, wood lignocellulosic sugars are shown to be
completely utilized
(fully depleted) in the culture solutions. Also, the efficiency of conversion
of hydrolysate
into biomass is measured to demonstrate the impact of process residuals, in
addition to the
sugars, for producing algal biomass and product. This is an essential feature
that must be
monitored to decide if a specific microbial bioconversion method warrants
implementation at
a mill biorefinery site.
Accordingly, for the purposes of this invention, "a wood processing operation"
refers
to an industry that processes wood for various uses, for example, printing and
writing paper
grades, various coated and uncoated specialties and paper grades, tissue and
toweling
products, paperboard, medical packaging, absorbent and air laid non-woven
products (such as
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diapers, hygiene, incontinence products), textile fibers, film, and sawn
timber. These
products utilize many types of woody biomass that may comprise but are not
limited to
Northern Softwood (for example Lodgepole Pine, White/Engelmann Spruce, Jack
Pine, Sitka
Spruce, Norway Spruce, and Black Spruce); Northern Hardwood (for example
Maple, Birch,
Poplar); Southern Softwood (for example Loblolly Pine, Shortleaf Pine);
Southern Hardwood
(for example Oak, Maple and Poplar). The other woody biomass in the supply
chain
comprises but is not limited to debarking residues, chip screening residues,
knots and pulp
fibers. The lignocellulosic biomass can also be a byproduct from the wood-
processing
operation. The associated mills can be of various types and can include
chemical pulp mills
(such as sulfate mills and sulfite mills) and chemical-mechanical pulp mills
(such as TM?
and CTMP mills). Additional embodiments of wood processing operations that
supply
biomass containing lignocellulose can be identified and used according to the
methods
described herein by a person of ordinary skill in the art and such embodiments
are within the
purview of the invention.
Thus, the invention provides improved methods for producing algal product from
woody feedstocks, particularly for methods that provide a means to produce
target products,
and preferred profiles, using obligate and facultative heterotrophs, with and
without
pigments, with greater yield and efficiency. The present invention meets this
need for this
non-commonly used wood-derived lignocellulosic hydrolysate feedstock with
exemplification for several algal products.
An embodiment of the invention provides processing a lignocellulosic biomass,
wherein the lignocellulosic biomass is raw material for a wood processing
operation. The
lignocellulosic biomass can be wood or byproduct from the wood-processing
operation.
The major chemical constituents of softwoods and hardwoods used in a wood
processing operation are shown in Table 1, with hardwood have much higher
hemicelluloses
and much lower lignin than softwood.
Softwood and hardwood as used herein refer to the physical structure and
makeup of
the wood, i.e., hardwoods is hard and durable; whereas, compared to hardwood,
softwood is
soft and workable. Hardwood typically comes from angiosperm - or flowering
plants - such
as oak, maple, or walnut, that are not monocots. Softwood typically comes from
gymnosperm trees, usually evergreen conifers, like pine or spruce.
Lignocellulose-derived process residuals from a typical softwood, Norway
Spruce
used in a wood processing operations is also shown in Table 1. However across
the various
wood species there can be a range of chemical composition values for both wood
and for bark
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as shown in Table 2. Various chemical compositions are shown in Table 3 for
some North
American woods. Specific chemical composition within a species for wood, bark
and
knotwood for Scots Pine, a softwood, is shown in Table 4.
Species variations among softwood lignins are relatively negligible in
contrast with
hardwood lignins. For the hemicelluloses, softwood and hardwoods are quite
different
chemically, as is known in the art. The different hemicellulosic
polysaccharides for the two
groups show various hydrolysis rates to produce different yield amounts of
degradation
sugars using the same process conditions. Norway Spruce typifies the
generalized softwood
profile. Upon degradation of Norway Spruce by pretreatment, numerous
categories of
constituents can result; most of the lignocellulose-derived inhibitors in the
process residuals
form when hemicelluloses and/or lignin are solubilized and degraded (Table 1).
In addition, new compounds are still being identified as inhibitors in
lignocellulosic
biomass hydrolysates. Some compounds function as co-inhibitors, producing
negative
synergistic effects (with no negative effect as individual compounds) that
affect longer lag
phase, slower growth, lower cell density, and lower product yield with lower
glucose
consumption (Zha et al. 2014).
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Table I. A generalized chemical composition of softwoods and hardwoods.
Cellulose Hemiceilulose
Softwoods* 43% , 28% _ 29%
Hardwoods 45% _ 34% 21%
nuGos,Accio.RitiEs
,,....."-- ANDDiSA.CCHARIDES.
Celiilloci? -
40X, ittxo/sr.
A5 h,_ ..,. :.1..., ' Lignin
swan
Extractives-----
= PMENYLIC -2% z,- ' . .........
CONIROUNOS Wade*
1 i:-:
1 Mammon
,,,,,,,"*. =!:"i , cuifulases
WttclialiC
C.C3erpour4i, foe
PINTO%
to, ÷,oenr.1,, , V, SUGARS =-'.--
13ENZO = FUFANS 7..
t.i.Pt,>C 41(4, mt141444,
CtUitiON.F.S
it.111.1rmt oc,41 rOrµI'L Nthl. .
Ic.1 . - = .. ..: ..= = . =.74,pi,. .Ai
IRONIC 4ØP.milvi..
. ..
. ..
ACIDS 1,100:,f40.4:4,,d . ..
. .=
.= Aict . .L.=:.:
,a,i lb = 0,1
" OC LI ,4 itl
ALIPHIAY IC #101,01411C. .
AUKHYDES CAN0OXYUCA003
* Norway Spruce typifies the generalized softwood profile. The hemicelluleses
and/or lignin
origin of many process residuals is shown here. Taken from Jonsson and Martin
(2016).
Table 2. Ranges for constituents by mass of lignin, polysaccharide, extractive
and ash in
woods and barks. See, world-wide website: carboleamlie/wood.php and USDA
(1971).
Component Softwoods Hardwoods
. Wood Bark Wood Bark
L. _____________________________ .
Lignins 25-30 40-55 18-25 40-50
Polysaccharides 66-72 30-48 74-80 32-45
Extractives 2-9 2-25 2-5 5-10
Ash 0.2-0.6 Up to 20 0.2-0.6 Up to 20
Bark as a woody biomass is quite heterogeneous and chemically complex.
Compared
to wood, bark has elevated levels of ash, lignin, and extractives and lower
levels of
polysaccharides. Extractives in bark are both much more abundant, more
variable, and also
unique than they are in wood. Bark extractives comprise lipophilic fractions
(e.g., fats,
waxes, terpenes and terpenoids, and higher aliphatic alcohols) and the more
abundant
SUBSTITUTE SHEET (RULE 26)
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hydrophilic fractions (e.g., phenolic constituents). Oligosaccharides include
about 60-70%
glucose, 5-15% xylose, 5-10% arabinose, and 3-4% each of galactose and
mannose, with
raffmose and stachyose present in minor amounts in bark (USDA 1971). Chemical
composition of knots and different fractions of wood is shown for Scots Pine
in Table 4. The
highest lignin content and extractives content (9%) was determined for
knotwood (32%).
Nevertheless, bark, knotwood, and the other wood fractions from Scots pine
that
differed significantly in chemical composition, were shown to be relatively
similar with
regard to susceptibility to pretreatment and enzymatic saccharification. The
most obvious
difference with regard to enzymatic saccharification was that without
pretreatment the bark
fraction was more susceptible than the other fractions. This similarity in
saccharification
outcome is considered favorable for the utilization of wood operation residues
from Scots
pine for bioconversion to fuels and to chemicals.
Table 3. Chemical composition of some North American woods. See, world-wide
website:
web.nchu.edu.tw/pweb/users/taiwanfir/lesson/9324.pdf
Percentage
Species Glucose Xylose Galactose Arabinose Mannose Uronics Acetyl .. Ugnin ..
Ash
Red Maple 46 19 0.6 0.5 2,4 3.5 3,8 24 0.2
Sugar Maple 52 15 <0.1 0.8 2,3 4.4 2.9 23 0.3
Yellow Birch 47 20 0.9 0.6 3.6 4,2 3.3 21 0.3
Paper Birch 43 26 0.6 0,5 1.8 4.6 4.4 19 0.2
Beech 46 19 1.2 0.5 2.1 4.8 3.9 22 0.4
Sweetgum 39 18 0.8 0.3 3.1 - -- 24 0.2
Quaking Aspen 49 17 2.0 0,5 2.1 4.3 3.7 21 0.4
Southern Red Oak 41 19 1.2 0.4 2.0 4.5 3.3 24 0.8
American Elm 52 12 0.9 0.6 2.4 3.6 3.9 24 0.3
Balsam Fir 46 6.4 1.0 0.5 12 3.4 1.5 29 0.2
White Spruce 45 9.1 1.2 1.5 11 3.6 1.3 27 0.3
Black Spruce 44 6.0 2.0 1.5 9.4 5.1 1.3 30 0.3
Jack Pine 46 7,1 1,4 1.4 10 3.9 1.2 29 0.2
Red Pine 42 9.3 1.8 2.4 7.4 6.0 1.2 29 0,4
Eastern White Pine 45 6.0 1,4 2.0 11 4.0 1.2 29 0.2
Loblolly Pine 45 6.8 2.3 1.7 11 3.8 1.1 28 0.3
Northern White Cedar 43 10.0 1.4 1.2 8.0 4.2 1.1 31
0.2
Eastern Hemlock 44 5.3 1.2 0.6 11 3.3 1.7 33 0.2
* Based on extractive-free wood.
Table 4. Chemical composition of fractions of Scots pine.
SUBSTITUTE SHEET (RULE 26)
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cont.. ___________________________ 1 %
/mann A.vM G,1a asgsa Maxim ANdli:k Mason Vag
Antd soluble Mat VOA Eacbwelftw ,844 Tuba
SD 3D 3D *SD $D ISD NSW. 1SD 3D .SD
$D
Juvenile 42.7.h 6.6 0.2.1
bearm008 3.019.1 7.1 0.1 3,9 '1 02 0,1 27,9103 1,640.1
29,9404 4.9:1115 0.1 100
Mauro 42.2k 5.34
hesectwond 1.840.1 3.01411 5.2 12.1 8.2 0.1 17.7111,1
1.4 1 0.1 293 4 0.2 4.440.6 Ill 911
hwenik 39.7 1 6.7 82
OPWOOd -t0.1 2.8.0,2 0.1 10.54 03 03 25.8
1.740.1 27.449.5 3Ø0,2 0.1 92
Malts 41,81 5.3 0.2
mymood .7 .13.1 2.3 0.1 1:6 14Ø112 al 28.910.2 I5 0.3 2S4
0.2 19103 0.1 97
41,81 .5,41 0.94
Mak 2440,2 2.849.2 2,0 11,7403 0.1 28.240.3
1.940.1 30.940.3 33 4 01 9.3 99
41.4 6.61 113
Top pacts 2,0.0,1 7.4 9.! :.4 L.4 8.1 ( 1 29.1 0.2
1.610.: 29.719.2 33.0,6 0,1 98
Kuore.I.Iod 2.2 0.1 4.14.03 38.24: 11.9 0.! 0.6 30.0 03 31.5 0.3 92
9.3 0.3 105
91 03
aYkid5sg 160 r aenodbm-k. M' 1 c11 mnd friandzed ddviadons bawd os du=
irplicatcs.
bldubdas =bins; gaindon, iducon, raannan, xyiro, tosaE =active% and isah.
An embodiment of the invention provides a method of treating lignocellulosic
biomass, for example, a biomass that is raw material for a wood processing
operation. The
method of treating a lignocellulosic biomass comprises the steps of:
a) providing a lignocellulosic biomass, wherein biomass comprises a
lignocellulosic
compound,
b) hydrolyzing the lignocellulosic biomass to produce a lignocellulosic
hydrolysate,
wherein the lignocellulosic hydrolysate comprises a simplified sugar produced
from at least a
portion of the lignocellulosic compound,
c) optionally, separating the lignocellulosic hydrolysate into a first portion
and a
second portion and treating the second portion of the lignocellulosic
hydrolysate to convert a
portion of the lignocellulosic compound and/or the simplified sugar to a non-
sugar agent;
d) mixing the treated second portion of the lignocellulosic hydrolysate
comprising the
non-sugar agent with the first portion of the lignocellulosic hydrolysate.
A further embodiment of the invention also provides a method of producing a
culture
medium for culturing a microbe to produce a product of interest. The method of
producing a
culture medium comprises the steps of:
a) providing a lignocellulosic biomass, wherein the lignocellulosic biomass
comprises
a lignocellulosic compound,
b) hydrolyzing the lignocellulosic biomass to produce a lignocellulosic
hydrolysate,
wherein the lignocellulosic hydrolysate comprises a simplified sugar produced
from at least a
portion of the lignocellulosic compound,
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c) optionally, separating the lignocellulosic hydrolysate into a first portion
and a
second portion and treating the second portion of the lignocellulosic
hydrolysate to convert a
portion of the lignocellulosic compound and/or the simplified sugar to a non-
sugar agent;
d) mixing the treated second portion of the lignocellulosic hydrolysate
comprising the
non-sugar agent with the first portion of the lignocellulosic hydrolysate,
e) producing a culture medium comprising the mixture obtained after step d).
An embodiment of the invention also provides a culture medium produced
according
to the method described above, which, as noted above, is referred to as "a
method of
producing a lignocellulosic hydrolysate containing medium."
In an embodiment, the step of hydrolysis is performed using a hydrolytic
enzyme,
preferably an enzyme that hydrolyses lignin, lignocellulose, or cellulose, for
example,
ligninase, lignocellulase, hemicellulose, or cellulase. Conditions appropriate
for an enzyme
used for the hydrolysis of lignin, lignocellulose, and cellulose are well
known in the art and
can be appropriate used by a skilled artisan.
The term "hydrolytic enzyme(s)" is meant to refer to enzymes that catalyze
hydrolysis
of biological materials such as cellulose. Hydrolytic enzymes include
"cellulases," which
catalyze the hydrolysis of cellulose to products such as glucose, cellobiose,
cello-
oligodextrins, and other cello-oligosaccharides. "Cellulase" is meant to be a
generic term
denoting a multienzyme complex or family, including exo-cellobiohydrolases
(CBH),
endoglucanases (EG), and 13-glucosidases (13G) that can be produced by a
number of plants
and microorganisms. Many crude cellulase extracts also include some
hemicellulases. The
process in accordance with embodiments of the invention may be carried out
with any type of
cellulase enzyme complex, regardless of their source; however, microbial
cellulases are
generally available at lower cost than those of plants. Among the most widely
studied,
characterized, and commercially produced cellulases are, e.g., those obtained
from fungi of
the genera Aspergillus, Humicola, and Trichoderma, and from the bacteria of
the genera
Bacillus and Thermobifida. Also, for example, cellulase produced by the
filamentous fungi
Trichoderma longibrachiatum includes at least two cellobiohydrolase enzymes
termed CBHI
and CBHII and at least 4 EG enzymes.
The non-sugar agent can be an organic acid such as acetic acid, propionic
acid, citric
acid, fumaric acid, glycolic acid, lactic acid, malic acid, pyruvic acid,
succinic acid,
glucuronic acid, galacturonic acid, and ferulic acid.
In some embodiments, the culture medium contains process residuals from the
wood
lignocellulosic hydrolysis process that comprise non-sugar agents. Use of
process residuals
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from the wood lignocellulosic hydrolysis process that comprise non-sugar
agents according
to the invention provides enhanced growth of the algal biomass relative to an
algal culture
control grown in a medium that lacks the non-sugar agents
An embodiment of the invention also provides a culture medium which contains
process residuals from the wood lignocellulosic hydrolysis process that
comprise a non-sugar
agent. In one embodiment, the non-sugar agent is an organic acid, an alcohol,
a
micronutrient, a salt, a saponifiable or fatty acid compound, a furfural,
process water, a
protein, or any combination thereof In a further embodiment, the organic acid
is acetic acid.
The acetic acid present in the culture medium can be produced by the
lignocellulosic
hydrolysis or by a microbial conversion solution wherein the microbe has
produced the acetic
acid from the lignocellulosic hydrolysate. In one embodiment, the acetic acid
is present with
at least one other fixed carbon source, for example, a sugar. In certain
embodiments, the
organic acid is propionic acid, citric acid, fumaric acid, glycolic acid,
lactic acid, malic acid,
pyruvic acid, succinic acid, glucuronic acid, galacturonic acid, or ferulic
acid.
The product of interest can be biomass or a product present in biomass.
Microalgae
are a valuable biocatalyst for the conversion of hydrolysates into compounds
of preferred
compositions, including for biomass, lipids, proteins, pigments, and biomass
containing
recombinant genes. Algal biomass and extracts from several different species
are edible and
used in nutritional supplementation or coloration with affirmed GRAS status in
the US.
Other biomass contains protein comprised of all the essential amino acids and
useful for
animal feed including aquatic species feed. Yet other biomass is oil-rich and
useful for
bioenergy or for fractionation for obtaining polyunsaturated fatty acids
(PUFAs), notably
nutritional fatty acids or those of value for chemical modification for
industrial purposes.
Lipids are a group of naturally occurring molecules that include fats, oils,
vitamins
(e.g., A, E, D, and K), triglycerides, diglycerides, monoglycerides, sterols,
waxes and
phospholipids. They have broad functionality. For
example, polar lipids, notably
phospholipids, can form the structural components of cell membranes. These are
effective
emulsifiers and emollients and thus useful in skin-penetrating carriers, food,
and beverage
preparations. Other lipids such as neutral lipids store energy within cells,
with industrial
applications for biofuels and chemical raw materials. Some lipids, called
omega-3, omega-6,
and omega-9 polyunsaturated fatty acids, are well known for application in
animal and fish
feed, food, nutritional supplements, and pharmaceutical products. This
includes but is not
limited to docosahexaenoic acid (DHA); eicosapentaenoic acid (EPA); omega-3
docosapentaenoic acid (DPA n-3); omega-6 arachidonic acid (ARA); and omega-6
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docosapentaenoic acid (DPA n-6). Omega-3, 6-, and 9- fatty acids can be medium
to long
chain carbon molecules for a variety of industrial and real-world
applications.
Another product of interest is microalgal biomass with biological pigments.
Numerous naturally pigmented compounds, called carotenoids and xanthophylls,
are used as
antioxidants, anti-inflammatories, antiapogenics, feed and food colorants, or
for extraction
into nutritional supplements. These include fl-Carotene, lutein, lycopene,
astaxanthin, and
fucoxanthin. Several carotogenic microalgae have been shown to be facultative
heterotrophs
for cultivation in the dark whereby carbon dioxide used during photosynthesis
as the carbon
growth source is substituted by some other carbon source dissolved in the
nutrient medium.
These may include Scenedesmus almeriensis, Chlorella zofingiensis,
Muriellopsis sp.,
Chlorella sorokiniana and Chlorella protothecoides as sources of lutein. US
Patent
Application Publication No. 20050214897 discloses a method for production of
astaxanthin
from Chlorella zofingiensis in dark heterotrophic cultures. US Provisional
Application Serial
No. 62/356,896 discloses heterotrophic biomass production of astaxanthin-
forming species of
the Chlamydomonodales family.
Other products of interest are exemplified elsewhere herein.
PCT Publication W02009035551 and US Patent Application Publication 2009064567
and the references cited therein, all of which are incorporated herein by
reference in their
entirety, provide organisms and some culture conditions of microbes rich in
these long-chain
fatty acids or other fatty acids. These microbes can be used in the methods
described herein.
Heretofore, the provision for rapid heterotrophic or mixotrophic cell growth
in wood
lignocellulosic simplified sugar with product formation suited for commercial
applications
has not been successful for any facultative or obligate heterotrophic
microalgae. An
embodiment of the invention provides a method that in effect enables
manufacturing biomass
from a cell of class Chlorophyceae, Bacillariophyceae, Trebouxiophyceae,
Euglenophyceae,
Peridinea, Dinophyceae or Labyrinthulomycetes, or a product of interest
produced by a cell
of any of those classes.
For example, a further embodiment of the invention provides a method for
synthesizing a product of interest using fermentation. In one embodiment, the
method
comprises the steps of:
a) providing a culture medium produced according to the method of producing a
medium containing lignocellulosic hydrolysate;
b) providing a microalgal cell that produces the product of interest;
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c) culturing the microalgal cell in the culture medium to produce a microalgal
culture
from the microalgal cell; and
d) purifying the product of interest from the microalgal culture.
In some embodiments, the cell is capable of depleting the sugar in the culture
medium. In some embodiments, a monoculture of the microalgal cell is capable
of utilizing
both C5 and C6 sugars.
Certain embodiments of the invention enable co-culture with different cell
types,
which can include different microalgal species that do not require organic
acids for
heterotrophy but can preferentially utilize, and thus mitigate, accumulation
of high levels of
ammonium or other metabolites for rapid vegetative growth. In some
embodiments, the co-
culture is capable of depleting the sugar in the culture medium. In certain
embodiments, the
co-culture is capable of utilizing both C5 and C6 sugars.
The new method additionally enables co-culture with different cell types that
can be a
microalgal species and a yeast species for complete utilization of pentose and
hexose sugars
for various wood-derived feedstocks. In some embodiments, the co-culture is
capable of
utilizing both C5 and C6 sugars.
The heterotrophic microalgal product, either extracted or the biomass, can be
used for
animal feed, human nutrition and nutritional supplements, personal care,
colorant, bioenergy,
or recombinant gene targets. By these means, the myriad of critical advantages
gained by
large-scale fermentative algal culture can be realized for this vast potential
supply of wood-
derived carbon feedstock for production of proteins, lipids, carotenoids,
recombinant gene
target, and other products.
The use of indoor fermentation vessels for heterotrophy as described herein
offers a
new solution for biosecurity for strains that are cultured phototrophically
and outdoors at
large volume. Use of fermentation tanks, especially tanks located indoors, can
simplify
regulatory approval of the industrial scale manufacture of recombinant
products.
In one embodiment of the invention, a microalgal cell is used to produce a
culture.
Nonlimiting examples of microalgae that can be used in accordance with the
present
invention include the following: Achnanthes orientalis, Agmenelluin,
Amphiprora hyaline,
Amphora cofieiformis, Amphora dehcatissima, Amphora delicatissima, Amphora
sp.,
Anahaena, Ankistrodesmus, Ankistrodesmus ,falcatus, Asteracys spp.,
Auxenochlorella
protothecoides, Boekelovia hooglandii, Borodinella sp., Botryococcus brciunii,
Botryococcus
,sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria,
Chaetoceros
Chaetoceros 171 uelleri, Chaetoceros rnuelleri subsalsum, Chaetoceros sp.,
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Chlamydomonas reinhardtii, Chlamydomonas dysosmos, Chlorella crnitrata,
Chlorella
antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulate,
Ch/ore/la
desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca,
Chlorella fusca var.
vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infirsionum
var. actophila,
Chlorella infusionum var. awcenophila, Chlorella kessleri, Chlorella
lobophora, Chlorella
luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis
var. lutescens,
Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella
nocturna, Chlorella
ova/is, Chlorella parva, Chlorella photophila, Chlorella pringsheimii,
Chlorella
protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis,
Chlorella
regularis var. minima, Chlorella regularis var. umbricata, Chlorella
reisiglii, Chlorella
saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella sauna,
Chlorella simplex,
Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella
stigmatophora,
Chlorella vanniellii, Chlorella vulgaris, Chlorella xanthella, Chlorella
zofingiensis,
Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum,
Chlorococcum sp.,
Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp.,
Crypthecodinium
cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana,
Cyclotella sp.,
Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella
granulate, Dunaliella
maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella
primolecta,
Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella
viridis, Dunaliella
tertiolecta, Eremosphaera viridis, Eremosphaera sp., Elhpsoidon sp., Euglena,
Franceia sp.,
Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp.,
Haematococcus
spp, Heterochlorella sp., Heterosigma akashiwo, Humidophila, Hymenomonas sp.,
Isochrysis
aft'. galbana, Isochrysis galbana, Lepocinclis, Marinichlorella kessleri,
Mayamaea sp.,
Mayamaea permitis var. pacifica, Micractinium sp., Monoraphidium minutum,
Monoraphidium sp., Nannochloris sp., Nannochloropsis oceania, Nannochloropsis
sauna,
Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula
pseudotenelloides,
Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp.,
Nephroselmis
sp., Nitzschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschia
dissipata,
Nitzschia fru.stulum, Nitzschia hcmtzschiana, Nitzschia inconspicua, Nitzschia
intermedia,
Nitzschia microcephala, Nitzschia pus/la, Nitzschia pusilla elliptica,
Nitzschia pusilla
monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis
parva, Oocystis
push/a, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria
subbrevis,
Ostreococcus sp., Parachlorella beijerinckii, Parachlorella kessleri,
Parachlorella sp.,
Pascheria acidophila, Pavlova sp., Phagus, Phormidium, Pinguiococcus
pyrenoidosus
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Plinymonas sp., Pleurockysis carterae, Plettrochrysis dentate, Pleurochrysis
sp., Prototheca
wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca
inorifi)rmis,
Prototheca zopJli, Pseudochlorella (viatica, Pyrambnonas sp., Pyrobotrys,
Rhodococcus
opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Scenedesmus obliquus,
Schizochytrium sp., Sellaphora sp., Spirogyra sp., Spirulina platensis,
Stichococcus sp.,
Synechococcus sp., T-Isochlysis, Tetraedron sp., Tetrasehnis sp., Tetraselmis
suecica,
Thalassiosiraweissflogii, Thraustochytrium sp., Ulkenia sp., and Viridiella
fridericiana.
In certain embodiments, the methods provided herein can be used for the
expression
of a recombinant protein or recombinant RNA by culturing a microalgal cell
expressing the
recombinant protein or recombinant RNA. The
microalgal cell can belong to:
Haematoccocus sp., for example, H. pluvialis; Chlamydomonas sp., for example,
Chlamydomonas reinhardtii; Scenedesmus sp., for example Scenedesmus obliquus.
Use of co-cultures in one method of the invention could be beneficial. The
cells for a
co-culture can be selected such that they require an alternative carbon or
nitrogen source that
can be withheld or supplied when needed to modify population dynamics
affecting target
product production in the co-culture. Accordingly, one embodiment of the
invention
provides a method of using lignocellulosic feedstock for co-cultivating two
cultures that are
both facultative heterotrophs belonging to the class Chlorophyceae or
Trebouxiophyceae.
Another embodiment provides a method for co-cultivating two cultures with one
being a
facultative heterotroph belonging to the class Trebouxiophyceae and the other
being an
obligate heterotroph yeast, Rhodotorula.
In some embodiments, the culture medium contains process residuals from the
wood
lignocellulosic hydrolysis process that comprise non-sugar agents. Use of
process residuals
from the wood lignocellulosic hydrolysis process that comprise non-sugar
agents according
to the invention provides enhanced growth of the algal biomass relative to an
algal culture
control grown in a medium that lacks the non-sugar agents. In certain
embodiments, culture
media for members of the genera Chlorella, Scenedesmus, Parachlorella,
Crypthecodinium,
and Schizochytrium comprises pulp and paper mill lignocellulosic hydrolysate
with simplified
sugars and one other non-sugar process residual also present in the
hydrolysate to provide
enhanced growth or faster fermentation cycle time.
In some embodiments, the culture medium contains process residuals from the
wood
lignocellulosic hydrolysis process that comprise non-sugar agents that are
organic acids.
In some embodiments, the culture medium contains acetic acid as part of the
lignocellulosic hydrolysate or as part of a microbial conversion solution
wherein the microbe
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has produced the acetic acid from the lignocellulosic hydrolysate. In one
embodiment, the
acetic acid is present with at least one other fixed carbon source, for
example, sugar. In a
further embodiment, the acetic acid is always with at least one other fixed
carbon source
which is a sugar.
In one embodiment, the wood lignocellulosic sugar stream provides a slip
stream that
is used to make organic acid by microbial conversion (bioconversion). The
lignocellulosic
sugar is conveniently provided in the same solution as the resulting organic
acid due to
incomplete utilization (incomplete bioconversion) by the converting microbe,
such as a
bacterium.
In another embodiment, the invention provides a culture medium comprising a
lignocellulosic sugar and a wood-derived organic acid, where the organic acid
is the preferred
fixed carbon source for one cell type and the sugar is the preferred fixed
carbon source for the
other cell type during heterotrophic or mixotrophic fermentation. In one
embodiment, the
algal culture medium can be supplemented with sugar from the lignocellulosic
hydrolysate
stream or from another sugar source. In one embodiment, lignocellulosic
hydrolysate stream
is used to stress an algal culture in the wood-derived organic acid medium.
Accordingly, the fixed carbon source used in the methods described herein can
be a
carboxylic acid, sugar acid, or chlorogenic acid. Non-limiting examples of a
fixed carbon
source include acetic, succinic, citric, fumaric, glycolic, malic, pyruvic,
glucunoric,
galacturonic, formic, levulinc, or proprionic acid. In certain embodiment, the
organic acid
used as a fixed carbon source can be derived from lignocellulosic biomass.
Additional
examples of a fixed carbon source are known to a person of ordinary skill in
the art and such
embodiments are within the purview of the invention.
In certain embodiments, the glucose to nitrogen ratio can be optimized to
provide the
best profile of a product.
In an even further embodiment, the invention provides a heterotrophic co-
cultivation
with at least one other microorganism. In an embodiment, the mutualism between
two
microalgal strains is described where accumulation of high levels of ammonium
(NH4+
NH3) or other metabolites that might otherwise inhibit cell division of one
strain is mitigated
by the other strain.
In certain embodiments, co-culture or co-cultivation is used as a strategy to
promote
proliferation of the target species. In a particular embodiment, co-culture is
between a strain
that requires organic acids as its fixed carbon source for heterotrophy and
another strain that
does not require organic acids for heterotrophy and can preferentially utilize
ammonium or
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the other metabolite such as ethanol, lactate, or formate that can accumulate
under low
oxygen.
The present invention further relates to generating and cultivating
microorganisms
suited for heterotrophically producing high yields of carotenoids for biomass
and products
containing said microorganisms or said carotenoids.
In certain embodiments, a culture medium for a member of the genus Mayamaea
comprises pulp and paper mill lignocellulosic hydrolysate with simplified
sugars and one
other non-sugar process residual also present in the hydrolysate. In one
embodiment the
microalgal cell of the genus Mayamaea expresses significantly reduced
fucoxanthin
pigmentation. Many diatoms are excellent sources of PUFAs and also
characterized by
containing fucoxanthin, which generally are extracted out with the lipids.
Thus it is preferred
to reduce pigmentation for facilitating the decolorization of the lipids.
In certain embodiments, heterotrophic cultivation of a genetically engineered
organism is described. In one embodiment, a culture medium, for mixotrophic or
heterotrophic fermentation of a member of the genus Chlamydomonas, consists of
pulp and
paper mill lignocellulosic hydrolysate that has been further processed by
microbial
conversion into a mixture comprising organic acid and residual unconverted
sugars.
In another embodiment, a culture medium for a member of the genus Scenedesmus
comprises pulp and paper mill lignocellulosic hydrolysate with simplified
sugars and one
other non-sugar process residual also present in the hydrolysate. In a
preferred embodiment
the microalgal cell expresses an added integrated heterologous gene.
The method described herein enables hydrolysate solutions to be easily
manageable,
provides a means to be cultivable with hardwood and softwood hydrolysates,
production all
during the year at a mill, and from which the desired product can be obtained
economically in
high yields.
The methods used in harvesting and further processing the biomass for
isolating a
product of interest are well known in the art. For example, some non-limiting
methods of
harvesting are centrifugation, flocculation, and filtration for dewatering.
Some methods of
extraction are in organic solvents, in edible oil, and by pressurized fluid
and gas. In certain
embodiments the heterotrophically produced biomass is used directly or as an
admixture in
animal and fish feed. For example astaxanthin-containing biomass is used for
fish feed, and
recombinant Chlamydomonas biomass is used in poultry feed. In other
embodiments the
pigments are extracted, for example astaxanthin is extracted as described in
the US patent
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6,022,701. A myriad of applications for astaxanthin include those described in
the art, for
example, in Ambati et al. 2014, Tables 4 and 5.
A further embodiment of the invention provides methods for improved
cultivation of
cells under mixotrophic conditions. In some cases it is desirable that light
be supplied to
increase the growth rate of the cells beyond that of heterotrophic conditions.
In H. pluvialis,
the specific growth rate under mixotrophic conditions is 2.5 fold higher than
the specific
growth rate under heterotrophic conditions. In C. reinhardtii, the specific
growth rate under
mixotrophic conditions is 1.8 fold higher than the specific growth rate under
heterotrophic
conditions. The combination of a lignocellulosic sugar and a non-sugar agent
in their
unpurified form in a hydrolysate described in the present invention are well
suited for
mixotrophic systems. This may be particularly advantageous for increasing
pigment
accumulation beyond the already high levels seen under heterotrophic
conditions, obtaining
higher biomass yields, or shortening a production cycle time. A further
advantage of
mixotrophic growth is that dissolved oxygen levels in the culture medium will
be easier to
maintain as the cells will be producing oxygen as they fix CO2 using light.
An embodiment of the invention provides methods of fermentation that do not
require
differentiation of the cultured cells for massive accumulation of a product of
interest.
Another embodiment of the invention also allows significant biomass,
carotenoid, and lipid
accumulation in the dark for measurably high specific growth and productivity
rates to enable
short cycle times. Thus, fermentation methods and cells are described that
provide higher
yields by a simple process in the dark without the need for cell
differentiation. The methods
of the invention provide culturing a microalgal cell, for example, a
genetically modified algal
cell, in a secure heterotrophic platform which transforms algal manufacturing
for significant
economic gain.
The methods of the invention provide:
1) higher production outputs, shorter incubation time;
2) simpler production logistics for feedstock by removing the step for fixed
carbon
purification;
3) altered product profiles;
4) faster industrial scaling by co-location at pulp and paper mills, with
existing
infrastructure useful for microbial fermentations;
5) use of a carbon feedstock with year round supply, at very large scale;
6) use of a carbon feedstock and process residuals with high efficiency of
bioconversion, and with complete utilization of the glucose and xylose in a
wood hydrolysate
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7) significantly increased inventory and access to much larger markets using
the
wood-derived feedstock; and
8) large-scale economic production of multiple species, including those used
as hosts
for recombinant molecules under GMP and regulatory restrictions.
Examples of general principles and methods for heterotrophic algae
cultivation, such
as establishing axenic cultures, using a seed train with a plurality of
passages prior to addition
of final inoculum, the design of the fermentors that prevent illumination or
add illumination
of the microalgae, and cultivation until harvest or partial harvest, are
described in the art, for
example, in US patent 8,278,090, which is incorporated herein by reference in
its entirety.
In particular embodiments, the inoculum added to the fermentor can be produced
by
cultivation of the microalgae in the dark for at least one passage prior to
its addition to the
fermentor, or by prior cultivation in the dark for a plurality of passages,
e.g., 2 passages, 3
passages, 4 passages, or 5 or more passages. In certain embodiments, after
cultivation of the
microalgae in the fermentor for a period of time in the dark, all or a portion
of the microalgae
can be transferred to a further fermentor vessel, where the microalgae can be
further cultured
for a period of time, wherein the further vessel prevents exposure of the
microalgae to light.
In practical terms, microalgae reported as having a mixotrophic capability,
for example,
various members of the Trebouxiophyceae, Bacillariophyceae, Eustigmatophyceae,
Prasinophyceae, are candidates for the practicing of the invention.
Harvest or separations, biomass processing, handling of intact biomass as a
product,
cellular lysis, product extraction, supercritical fluid processing, or other
isolation and
purification of products may be done by using any methodology known to a
person skilled in
the art. Non-limiting examples of such techniques are described, for example,
in US patents
8,278,090 and 7,329,789, both of which are incorporated by reference. Non-
limiting
examples of product recovery include the separating different target compounds
by use of a
fractional distillation column. Further non-limiting examples for
concentration, drying,
powdering, grinding in preparation for extraction or use as a biomass for
animal and fish
feed, are described, for example, in US patent 6,022,701 and EU Patent
Application
Publication No. EP1501937, both of which are incorporated herein by reference.
US Patent
Application Publication No. 20120171733 describes various means for cell lysis
that are
incorporated herein by reference.
US Patent Application Publication No. 20090214475, which is incorporated
herein by
reference, describes soft wall mutant strains of Haematococcus pluvialis for
improving the
extractability and bioavailability of natural astaxanthin, and their use in
animal feed, human
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dietary supplements, pharmaceuticals, and foods. This can apply to other non-
cyst forming
species that product astaxanthin such as Monoraphidium, Scenedesmus and
Chlorella.
Typical microbial growth curves or growth cycles are seen using a fermentor.
As an
example, an inoculum of cells when introduced into a medium is followed by a
lag period
before cell growth or division begins. Following the lag period, the growth
rate increases
steadily and enters the log, or exponential, phase. The exponential phase is
followed by
slowing of growth (cell division) due to nutrient depletion and/or increases
in inhibitory
substances. When growth stops the cells enter a stationary phase or steady
state.
In certain embodiments of the present invention it is desirable to
heterotrophically
cultivate a genetically engineered microorganism, for example using synthetic
biology where
pathways are introduced or templates for hairpin nucleic acids are introduced,
to enhance
traits such as the production of a recombinant molecule, to modify the
properties or
proportions of components generated by the microorganism, or to improve or
provide de
novo growth characteristics.
Genetic engineering of Haematococcus spp. (Sharon-Gojman et at. 2015),
Chlamydomonas spp. (Lauersen et at. 2013; Scaife et at. 2015; Scranton et at.
2015),
Scenedesmus obliquus (Guo et at. 2013), Prototheca and Chlorella are well
documented and
incorporated by reference herein.
As such, a method for synthesizing a product of interest is provided herein.
The
method comprises the steps of:
providing a culture medium comprising wood-derived lignocellulosic simplified
sugar
in the presence of a non-sugar agent, wherein the non-sugar agent is a process
residual of
wood lignocellulose hydrolysis or an organic acid solution obtained by
microbial conversion
of wood-derived lignocellulosic sugar;
providing a microalgal cell that produces the product of interest;
culturing the microalgal cell in the culture medium to produce a microalgal
culture
from the microalgal cell; and
purifying the product of interest from the microalgal cells.
The product of interest can be a microalgal biomass comprising the microalgal
cells,
lipid, protein, amino acid, recombinant molecule, or a pigment.
The pigment can be a carotenoid that is an astaxanthin.
The protein can be a total crude protein or a peptide fragment of a protein.
The lipid can be a total crude lipid, a phospholipid, a fatty acid, or a long
carbon chain
polyunsaturated fatty acid.
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The recombinant molecule of interest can be a heterologous protein or a
nucleic acid.
Also, the methods described herein provide for the heterotrophic growth and
synthesis
of the product of interest occurs during the step of culturing, for example,
vegetative growth
under nutrient replete conditions for phospholipid and protein production, and
stationary
growth for polyunsaturated fatty acids, and wherein the step of culturing is
performed under a
fed-batch fermentation.
As mentioned above, unlike conventional methods where the product of interest
is
synthesized using purified sugars or organic acids, the methods provided
herein permit the
synthesis of a product of interest using unpurified sugars or organic acids to
simplify the
feedstock processing steps.
As mentioned above, unlike conventional methods where the product of interest
is
synthesized using culture medium with the sequential additions of different
purified sugars
(e.g., hexose followed by pentose), or sequential additions purified sugar and
then organic
acid, the methods provided herein permit the synthesis of a product of
interest under
simultaneous supply of the compounds for the cell culture.
Because these algae are produced in closed culture systems to exclude
contamination,
the final biomass is of high quality suited for a variety of novel animal and
human uses. The
closed fermentation systems also offer large quantities at lower cost, being
produced at higher
densities and faster growth rates within a short cycle time of merely days. As
the carbon
feedstock for the fermentations are sourced from wood byproducts of vast
supply compared
to the seasonal grasses or other agricultural wastes, the algal products can
address markets of
high volume much more readily than the other feedstocks.
In certain embodiments, the product of interest is altered in component
composition,
proportion, or temporal expression as compared to a control, wherein said
control is a product
of interest produced by culturing a microalgal cell expressing said product of
interest in
culture medium comprising the simplified sugar but not containing the non-
sugar agent.
In certain embodiments, the product of interest is altered in component
composition,
proportion, or temporal expression as compared to a control, wherein said
control is a product
of interest produced by culturing a microalgal or microbial cell expressing
said product of
interest in culture medium comprising non-lignocellulosic sugars.
In certain embodiments of the culture method of the invention, the product
compositional analysis differs substantially from that seen by conventional
methods.
In certain embodiments of the culture method of the invention, without yet
undergoing optimization, the product compositional analysis can be as good as
or better than
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the best composition achieved with conventional methods.
In certain embodiments of the culture method of the invention, on a volume
basis, the
dry cell weight of the microalgae is greater than the dry cell weight of the
same strain of
microalgae cultured with a purified fixed carbon source that would require
further processing
steps to obtain and with all other culture conditions being the same. The dry
cell weight of
the microalgae grown using the culture medium of the invention can exceed the
dry cell
weight of microalgae grown using the same hexose and pentose source by at
least: about
40%, about 80%, about 100%, about 120% or more, or by an amount within any
range having
any of these values as endpoints.
The step of isolating and purifying the product of interest may comprise one
or more
steps of drying, grinding, lysing, or extracting the microalgal cell.
The step of culturing can be performed under mixotrophic conditions, at least
for a
portion of the culturing step.
The following examples are provided to describe the invention in further
detail.
These examples serve as illustrations and are not intended to limit the
invention.
EXAMPLE 1 ¨ WOOD ITYDROLYSATE FROM PULP AND PAPER MILL MATERIAL,
MICROBIAL SPECIES AND FERMENTATION CONDITIONS
Enzymatic 11,,drolysates of various compositions are produced courtesy of
Cellulose
Sciences International (Madison, WI) and Domtar International according to
U.S.
Patent 8,617,851 from various woody biomass, supplied by Domtar International,
subjected
to alkali plus co-solvent pre-treatment (Table 5). The enzymes product, used
according to
manufacturer's direction, was Cellie Ctec2 (Novozymes) that is a blend of
cellulases, beta-
glucosidases, and hemicellulase. Incubation was 72 hours with agitation, 50 C,
solids
loading of 2%, followed by filtration through a 10 kD filter to remove the
enzymes.
Lignocellulosic hydrolysates (Figure 1, [10]) from softwood and hardwood were
prepared
and analyzed. The algae strains selected for testing are based on their
potential biomass
applications for biofuels (lipids), feed (whole biomass, protein and lipids),
and specialty
products (colorants, nutritional lipids, emulsifying lipids) and capable of
heterotrophic or
mixotrophic growth. These include Hawaii-collected Ch/ore/la and Scenedestnus
identified
at the species level based on 18S sequence DNA sequencing, as described in
Kuehnle et al.
2015: KAS908 is 100% identical to Chlorella sorokiniana; KAS740 is 100%
identical to
Scenedestnus artnatus. Other non-limiting strains are listed elsewhere in the
examples.
Cultures were screened previously for their ability to grow on sugars and were
adapted for
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heterotrophic growth in modified F/2-Si (Si-free) fresh water medium
containing 18 g/L
glucose plus 1.8 g/L yeast extract (YE). Basic recipes of F/2 and F media
(detailed in
Guillard 1975; Guillard 1962), contain all the nutrients essential for growth
of many fresh
water microalgae and are easily modified by omission of seawater and
silicates. Use of this
medium is not limiting for the purposes of this invention. The pH of the
hydrolysates ranges
from 4.9 to 5.5, thus the pH of each medium containing wood hydrolysate is
adjusted to pH
7.0 using 1M TRIS-HC1 (pH 8.0) prior to inoculation. Wood hydrolysates are
initially tested
for growth at small scale and the wood hydrolysate concentration with highest
growth for
each strain was identified. Briefly, heterotrophically adapted KAS908 and
KAS740 are
grown in 96-well plates on an orbital shaker 100 rpm using wood hydrolysate
standardized to
18 g/L and 9 g/L total sugars along with the components that comprise modified
F/2-Si fresh
water plus YE medium, 26 C. These strains are further grown in 50 ml medium in
250 ml
shake flasks on an orbital shaker 100 rpm at suitable wood hydrolysate
concentrations found
during small-scale tests. Growth is monitored daily by measuring 0D750.
For Crypthecodinium cohnii (ATCC 307727; KAS1701) cells of the obligate
heterotrophic DHA producer are grown in medium with 20% or 40% BSP hydrolysate
(9 g/L
or 18 g/L total sugars), 1.8 g/L yeast extract (Difco, Sigma-Aldrich), and 60%
seawater
(equivalent to 21 g/L sea salt), pH 6.5. KAS1701 is cultured in 50 ml medium
in 250 ml
shake flasks on an orbital shaker 100 rpm at 26 C in the dark. For
Schizockytrium limacinurn
SRI (ATCC YA-1; KAS 1707 )_ cells of the obligate heterotrophic DHA producer
are
cultured (26 C in the dark at 100 rpm) and adapted to 1/2 strength seawater
(17.5 g/L Instant
Ocean) medium containing 25 g/L glucose supplemented with yeast extract, trace
elements,
and vitamins as described (Ren et al. 2009). Then 50 mL of log phase culture
is sub-cultured
to a 450 mL volume SPBK hydrolysate diluted such that total sugars starts at
25 g/L (also
contains 2.3 g/L acetic acid) plus nutrients in 1 L flasks.
Another strain tested is Rhodotorula glutinis (ATCC 2527; KAS1101) a red yeast
with high protein and oil, capable of synthesizing 13-carotene, torulene, and
torularhodin, of
interest as natural food colorants. It has shown synergistic effects when co-
cultivated with
Chlorella for increased biomass yield. Rhodotorula is maintained in YPD medium
comprised
of 10 g/L yeast extract (AMRESCO, VAVR), 20 g/L peptone (BD Bacto Peptone,
Fisher
Scientific), and 20 g/L glucose (Sigma-Aldrich). Other culture media, for
growth
comparisons, comprised 3% to 60% SHC hydrolysate with 10 gll yeast extract and
20 g/L
peptone (YP), with the resulting glucose concentrations: 5mM (2.7 g/L
glucose), 25 mM
glucose (4.5 g/L), 50 mM (9 g/L) and 100 mM (18 WO glucose.
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Table 5. Composition of Softwood and Hardwood Enzymatic Hydrolysates.
Total
Total process
Hydrolysate from
sugars % C6 % C5 residuals % Organic acids
Wood Alcohol
(g/L) (g/1-)
Acetic Lactic
Glu Man Gal Xyl Ara Ethanol
Acid acid
Hardwood HWD
18.68 60.52 1.51 1.79 35.60 0.63 5.11 2.15 52.52 45.32
(similar to SHC)
SPBK (Southern Pine
87.87 89.44 0.17 2.04 6.83 ND* 8.09 100 ND ND
Bleached Kraft)
SPFC (Southern Pine
21.51 62.00 18.14 0.78 18.44 0.65 3.67 50.16 49.94 ND
Finer Chips)
B SP (Bleached
44.33 90.91 ND ND 9.09 ND Not Provided
southern Pine)
SHC (Southern
27.50 56.96 0.69 13.02 29.33 ND ND ND ND ND
Hardwood Chips)
Hydrolysates profile: pH: 5.5
* ND: not detected.
Heterotrophic growth experiments are performed in the dark for wood
hydrolysates at
a larger scale using a 10-L BioFlo110 fermentor (New Brunswick Scientific,
Enfield CT) and
pre-established batch fermentation conditions of T =30 C, pH= 7.0, agitation =
300 rpm, DO
= 100%, and air = 7 L/min. Briefly, KA5908 is inoculated to a density of 2 g/L
in fresh
water medium, equivalent to 2x the concentration of F/2 medium, comprised of
wood
hydrolysates standardized to 18 g/L total sugars and the components of F
medium (0.2 g/L
Cell-HI F2P, Varicon Aqua Solutions, Worchestershire UK) plus 1.8 g/L yeast
extract.
Samples are collected every 24 hours for five days and analyzed for biomass
growth
measurement (dry weight), as well as for glucose and xylose utilization
through HPLC.
Culture samples in 25-mL quantities are collected and immediately centrifuged
at 3,000 rpm.
The supernatant from each sample is analyzed for glucose and xylose by HPLC
using a
Waters 2695 Alliance Separations module with a Rezex RPM-Monosaccharide Pb+2
(8%)
column (Phenomenex, Torrance, CA, USA) and a 2416 refractive index detector
(Waters
Corp., Milford, MA). Samples not immediately analyzed are stored at -20 C
until further use.
The system is run isocratically with deionized ultra-pure water. The injection
volume is 40
pL/min with a 20 min run time at 85 C.
Nitrate concentration is monitored qualitatively using a nitrate test kit
(Aquarium
Pharmaceuticals, Chalfont, PA). As positive controls and to establish baseline
kinetics,
fermentation using mixed C5 and C6 model sugars is also performed. In some
cases,
KAS908 is grown in F medium (modified for fresh water) containing 16.34 g/L
glucose and
1.66 g/L xylose plus 1.8 g/L YE to mimic the corresponding hydrolysate from a
first batch of
Bleached Southern Pine and grown under the same batch fermentation conditions
for five
days. BSP is identified as similar to SPBK by the supplier of the hydrolysate,
and made
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available in a subsequent preparation for additional larger scale experiments.
Biomass
productivities (g/L/day) and biomass yield on sugar (g total biomass/g sugar
utilized) are
calculated. Additional analytical methods utilized are described in the other
examples.
Cells from the 10 L volume of KAS908 fermentation culture can be used to
directly
seed a 80 L volume (10 L culture + 70 L fermentor heterotrophic media in an
Eppendorf
BioFlo 610 fermentor). The 80 L culture is fed nutrients using automated
peristaltic pumps
using BioCommand software and pH is maintained at 7.5 with 0.1 M NaOH and 0.2
M
H3PO4 as needed. The sparged air at 50-100 LPM and Rushton blade agitation to
350 rpm or
higher are controlled by a cascade and are increased as dissolved oxygen in
the system drops
below 50%. By this method the resulting biomass (16 g/L from an initial 0.2
g/L) is
produced over 96 hours that includes no lag phase and a 72-hour extended
logarithmic phase
of high specific growth of 1.4/day. For this and other species, it is
understood that scaling
from about 100 L to 1000 L to 100,000 L vessels and such can proceed using the
basic
conditions modified for mass balance, aeration, viscosity and cycle time as is
known in the
art.
The availability of differing preparations of feedstock informs a strategy for
the
carbon feed during the fermentation cycle, as the microalgal density increases
and
fermentation reactor capacity becomes more limiting; and for the choice of
microalgae and
co-cultivation option (if it prefers wood-derived 2-, 3-, 5-, and 6-carbon
feedstocks derived
from lignocellulosic biomass). The production volume is comprised of
relatively dilute
hydrolysate at the outset. As the culture growth actively increases, the
carbon is
proportionally supplied from conditioned, concentrated hydrolysate stream with
minimal
impact on working volume. In general terms, a concentrated feedstock
facilitates high
microalgal cell densities with minimal impact on working volume. This is
followed by a
finishing stage for the product of interest, as is known in the art. For
example, N stress or
cold stress, are used to promote carotenogenesis (for pigment accumulation) or
lipogenesis
(such as for omega 3-, 6- and 9-fatty acids accumulation), as shown in
subsequent examples
with several species and co-cultures. It is also understood that strains can
be selected for
improved product yield from populations cultured on wood hydrolysates, such as
from
various sources and concentrations, for increased productivities over time.
EXAMPLE 2¨ BIOMASS PRODUCTION ON WOOD HYDROLYSATES
Multiwell plates are used as an initial screening tool to determine the
capability of
microalgal cultures to grow in the dark on wood hydrolysates from pine
softwood, southern
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hardwoods and northern hardwoods. Surprisingly, all wood enzymatic
hydrolysates tested
support growth and biomass production of microalgae, though performance varies
with each
type of hydrolysate. For example, the three wood hydrolysates designated SHC,
SPBK, and
SPFC (Table 5), standardized to 18 g/L total sugars, show different growth
profiles for
Chlorella KAS908, with one hydrolysate (SPFC) being inhibitory for the first
four days
(Figure 2). During this period, culture using SPFC in the dark shows nominal
growth
(0D750 between 0 and 0.1) similar to the negative control in the dark using
F/2 with yeast
extract and no added sugars or hydrolysate (0D750 between 0 and 0.1), while
the growth of
positive controls on 9 g/L glucose and 18 g/L glucose reaches 0D750 above 0.3
by day 3.
Surprisingly, softwood and hardwood hydrolysates produce similar performance.
The
softwood (Southern Pine Bleached Kraft) yields active growth similar to
hardwood (Southern
Hardwood Chips), to reach only slightly less biomass yield by the fourth day
although it has a
longer growth lag for the first two to three days (Figure 2, showing day 0-day
4 growth).
Unexpectedly, onset of growth of KAS908 on Southern Pine Finer Chips (SPFC) is
evident
by Day 7, indicated by an increase in 0D750 from less than 0.1 on day 4 to
about 0.25 on day
7, whereas the negative control continued to show no growth as expected (0D750
less than
0.1); the positive control on 18 g/L glucose neared 0D750 of 0.4 by day 4.
These data
indicate a strategy of acclimation to certain hydrolysates to mitigate
inhibition or possible
inhibition by the process residuals, enabling use of higher amounts or
concentrations of
hydrolysates.
Surprisingly, Scenedesmus KA5740 cells can utilize process residuals. Using
KA5740 grown in flasks, use of SPFC corresponding to 9 g/L total sugars grows
better (60%
higher OD value at time of glucose depletion) than in medium containing 9 g/L
glucose alone
based on Student's t means testing (p = 0.02; Figure 3). Process residuals of
Southern Pine
Finer Chips contain two organic acids, acetic acid and lactic acid, while
Southern Pine
Bleached Kraft contains acetic acid and no lactic acid.
A red yeast, Rhodotorula glutinis, KAS1101 is grown in 96-well plates using
various
concentrations of SHC hydrolysates to compare with YPD medium with 20 g/L
glucose. As
shown in Figure 4, KAS1101 growth is uninhibited in the medium employing the
highest
amount tested of 60% SHC. It shows the same growth as the control YPD medium
on Day 2
and superior growth as the control by Day 4, with extended biomass yield for
one additional
day based on Student's t means testing (p =0.006; Figure 4) due to the process
residuals.
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A simple screen for relative growth patterns of microalgal species such as
described
here can be used to assist mills, which may be limited to producing a
particular wood
hydrolysate based on the mill products. Depending on their target products of
choice, the
mill may decide for conversion of a slipstream of hydrolysate into a second
carbon feedstock
(Figure 1, Y), such as into acetic acid, to then support microalgal
bioconversion using species
that favor organic acid as the primary fixed carbon source. See Example 5.
EXAMPLE 3 ¨ BIOMASS PRODUCT, SOME EXTRACTIVES, AND SUGAR
CONVERSION EFFICIENCY USING WOOD HYDROLYSATES
This example demonstrates higher biomass productivities on wood hydrolysate
than
on model sugars and higher than expected efficiency of bioconversion. Growth
of Chlorella
KAS908 in a medium based on softwood hydrolysate, Bleached Southern Pine (BSP
with
2F+ 1.8 g/L YE) hydrolysate, is compared to that in a medium containing an
equivalent
mixture of C5 and C6 model sugars (16.34 g/L glucose and 1.66 g/L xylose)
using a 7-L dark
stirred fermentor. Surprisingly, the wood hydrolysate with monosaccharides and
process
residuals outperform the model sugars alone, with a 1.6-fold (160%) higher
biomass
productivity of 2.87 g/L/day compared to 1.7 g/L/day for the control
Chlorella. KA5908
utilizes the glucose and xylose in series during dark fermentation, as shown
by a decrease and
eventual complete depletion of both sugars in the culture medium containing
wood
hydrolysates (Figure 5a), a feature mimicked during growth on model sugars
(Figure 5b).
Higher biomass production for both DHA-producing C. cohnii KAS1701 and
Schizochytrium KAS1707 is observed using hydrolysate compared with using model
sugar in
batch fermentation flasks per conditions in Example 1. KAS1701 grown in BSP
wood
derivative, corresponding to 9 g/L total sugars plus 1.8 g/L YE, shows rapid
increase in
0D750 from 0.5 to 4 on day 2 and 1.4 times higher yield than on pure glucose
(0D750 from
0.5 to 3) before reaching glucose depletion. Lipid and fatty acid analysis
indicates a lipid
content of 13% DW, with DHA comprising 30% of the lipid fraction for 4% DW.
Much
higher productivities are obtained when supplied with non-limiting feedstock
in fed-batch
mode and sufficient aeration. Switching to distillation-purified
ligoncellulosic acetic acid
(such as used in Example 5) also yields DHA, at 9% DW. For KAS1707, the
control flask
(glucose as only carbon source) and the SPBK flask the initial biomass of 0.5
g/L grows to 8
g/L and 9.2 g/L, respectively, in 72 hours with a maximum specific growth rate
of 0.95/day
and 1.1/day respectively. The cells are allowed to accumulate lipids for an
additional 24
hours and are harvested at 96 hours after inoculation. Volumetrically the SPBK
grown
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biomass contains 1.84 g/L (20% of the total biomass) total fatty acids and
0.46 g/L (5.0% of
the total biomass) of the fatty acid DHA. The control flask contains 1.44 g/L
(18% of total
biomass) total fatty acids and 0.37 g/L (4.6% of the total biomass) of the
fatty acid DHA.
The lipids are 1.1 times higher and the DHA is 1.08 times higher in the
presence of process
residuals from the softwood hydrolysate. With no carbon or nitrogen limitation
for the
purposes of reaching higher densities on SPBK concentrated to allow 140 g
accumulative
hexose during the course of fed-batch fermentation, cultures reach 70 g/L
biomass with 19
g/L (27% of total biomass) fatty acids. Higher biomass productivities
translate to increased
biomass product yield and shorter fermentation cycle times. Even higher final
omega-3 fatty
acids can result under nitrogen deficiency.
Biomass yield on sugar consumed (dry weight of biomass produced per gram of
sugar
utilized) is also determined, as a parameter useful in calculating overall
process efficiency
and biomass production cost. Results show that sugar utilization of
microalgae, using
different hydrolysate streams, varies with the composition and impurities
present in them.
Surprisingly, a high bioconversion ratio of 1.15:1 biomass produced per gram
of sugar
utilized, as measured by HPLC, is obtained for KAS908 grown in the hardwood
hydrolysate,
SHC. This exceeds the theoretical biomass yield per gram sugar utilized of
0.5:1 reported for
protein-rich algal biomass ideal for animal feed, and is attributed to the
assimilation of
process residuals in the hydrolysate; the hydrolysate glucose and xylose are
completely
depleted. The hardwood preparation has a relatively high ethanol content,
along with several
organic acids, and hydrolysate is known to contain furfurals. Also, BSP
hydrolysate gave a
0.45:1 ratio, close to the theoretical biomass yield per sugar utilized. The
development of this
method that proves suitable for microbial growth using cellulosic hydrolysates
from
softwoods, especially from Southern Pine, known for their unique toxic
fermentation
inhibitors, is beneficial to help advance implementation of the pulp and paper
mill biorefinery
concept.
These outcomes using the method of the invention show a high compatibility of
different algal genera for heterotrophic growth on wood hydrolysates and the
ability to scale-
up. This is required in order to develop process economics of using microalgae
for target
products, such as protein, lipids, and pigment production; to select host
strains for
recombinant product production that will be compatible with a certain mill's
lignocellulosic
feedstock; as well as to contribute to the potential of establishing
integrated biorefineries for
the pulp and paper industry. Once the target algal products are identified for
a mill, the algal
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strain can be optimized in tandem with the sugar and nutrient feeds as well as
operational
conditions. Some examples of other target algal products and alterations
follow.
EXAMPLE 4¨ ALGAL BIOMASS QUALITY FOR VALUE-ADDED PRODUCTS
In the next two examples, production of four different product classes, are
exemplified through the use of microalgae cultured in wood hydrolysates. These
comprise
lipids, protein, pigments, and recombinant product. It is understood that
these are non-
limiting examples, and that the production process can be optimized for each
cell type to
provide a preferred duration of the production cycle and preferred culture
conditions
throughout the fermentation to achieve the desired product formation.
A glucose:nitrogen ratio screening is performed to determine preferred ratios
for
improved quality of algal biomass for value-added products. Heterotrophically
acclimated
KAS908 cultures are grown in shake-flasks in a medium (2F + YE) with the
following
glucose:nitrogen ratios (w/w): 1:1, 3:1, 4:1, 5:1, 6:1, 9:1 and 13:1. On day
3, the medium
with 13:1 ratio gave the highest biomass density by (0D750 of 1.2) and the
medium on 1:1
ratio gave the lowest biomass density (0D750 of 0.4). Total crude fat is
determined by acid
hydrolysis/ petroleum ether method (AOAC 954.02 by New Jersey Feed Labs, NJ)
and
expressed as a ratio per total soluble proteins. Alternatively, lipids are
extracted and assayed
in algal cells using modified sulfo-phospho-vanillin methods (Cell Biolabs
Lipid Extraction
and Quantification Kits, San Diego, CA) following manufacturer instructions.
Protein is
extracted using a modified standard method for algae by Rausch (1981) and
quantified using
the Bradford reagent (ThermoFisher), with absorbance measured at 595nm using a
GENESYS 10S UV-VIS spectrophotometer. Relative amino acids are determined by
AOAC
994.12 and 985.28 by New Jersey Feed Labs. Analysis of phospholipids is by
thin layer
chromatography (TLC). Biomass was extracted in 2:1 chloroform:methanol (20 uL
per mg
biomass) and volumes from extractions were loaded onto TLC lanes after being
normalized
on a lyophilized-dry weight basis. Plates (Sigma-Aldrich TLC plates silica gel
matrix, Sigma-
Aldrich Co.) are run in a TLC chamber with chloroform:methanol:water 65:25:4
for 20
minutes, dried for 5 minutes, then sprayed with molybdenum blue spray reagent
(Sigma-
Aldrich Co.) and developed for 1 hour.
For pigment analysis, dewatered samples are pelleted by centrifugation at 3000
g for 5
minutes, frozen at -80 C, and freeze dried to determine dry weight. Pigments
are extracted
from ground freeze-dried biomass with 50 ILIL of acetone per mg of biomass for
5 minutes at
room temperature. For astaxanthin, mean pigment in acetone is determined by
calibrated
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spectrometry using the A476 absorbance adjusted by the extinction coefficient
of astaxanthin
(217) and proportion of total carotenoid that is astaxanthin in the vegetative
cell type (75%).
Constitutive (i.e., from the growth phase, non-stationary) protein and lipid
ratios are
compared between biomass grown in 40% BSP hydrolysate with F nutrients and the
biomass
grown in the dark in F medium with equivalent amounts of 18 g/L total sugars
(glucose +
xylose basis). Biomass derived from the hydrolysate culture contains an
altered protein to
lipid ratio of 1.8:1 compared to 3.4:1 for the heterotrophic control on
glucose and xylose
sugars alone. A more lipid-rich biomass on hydrolysate- under the conditions
used- is
advantageous for products of extracted oils such as for omega-3 fatty acids,
EPA and DHA,
and for phospholipids. Addition of organic acid-rich hydrolysate or wood-
derived organic
acids (Figure 1 [30]) can further increase the lipid content of the biomass of
KAS908 in F
with 18 g/L total sugars when under nitrogen-deplete conditions. Regarding
altered profile of
amino acids compared on a protein basis, biomass from the BSP hydrolysate has
almost
140% higher methionine + lysine than the control biomass, and 50% lower
valine. This can
benefit poultry feeds, formulated on amino acid requirements, to which
methionine + lysine
is normally added. Dried whole algal biomass as a feed ingredient delivering
protein and
dried lipid (to replace a certain amount of oil added in the broiler diet)
renders an improved
quality pellet for feed formulators. In the case of Chlorella KAS908 grown
under these
conditions, biomass from hydrolysate shows slightly lower linoleic acid
content than the
control biomass (37% vs 39% relative basis), with lowered levels preferred for
broiler
performance. Results also show altered alpha-linolenic acid, with a decrease
from 2.7% to
1.9% relative basis; ALA is added to animal feed to stimulate physiological
functions.
Qualitative results from TLC indicate that biomass grown for three days on the
4:1
ratio (Glucose:Nitrogen) gave the most intense bands for both PC
(phosphatidylcholine) and
PE (phosphatidyl ethanol ami ne) (Figure 6). This is validated at fermentor
scale carried out
using the previously determined growth parameters (temperature = 30 C, pH =
7.0, D02=
100%, agitation= 300 rpm, air = 3.0 Umin). KAS908 is grown in 2F medium
containing a
4:1 ratio of glucose to nitrogen (e.g., 3 gIL glucose to 0.736 g nitrogen).
Nitrogen sources are
YE and Cell Hi (0,7 giL YE, 0.3 g11_, Cell Hi). A TLC chromatogram indicates
that KAS908
grown in the dark for 3 days in a medium with 4:1 ratio of glucose to nitrogen
supports
higher production of phospholipids relative to the reference medium 2F +36 glL
glucose
(Figure 6, lane 3). This is compared to a 6-L dark fermentation on 40%
Bleached Southern
Pine wood hydrolysate (BSP) at 18 g/L total sugars. KAS908 biomass grown with
BSP
hydrolysate (Figure 6, lane 11) yields only slightly lower TLC band
intensities for
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phospholipids PC and PE than the best performing biomass grown on 4:1 glucose
to nitrogen
ratio medium (Figure 6, lane 9). Biomass grown at higher hexose and pentose
concentration
(19.43 g/L glucose and 8.06 g/L xylose in 2F, Figure 6, lane 10) has inferior
yields, similar to
2F + 36 g/1 glucose (Figure 6, lane 12). This shows that wood hydrolysate,
specifically of
Southern Pine, can be used directly (after dilution) for obtaining, for
example, a target lipid
product, with yields under unoptimized conditions using hydrolysate
approaching the highest
yields achieved under optimized culture using control medium without
hydrolysates and
without process residuals.
EXAMPLE 5 ¨ ALGAL PRODUCTS USING WOOD LIGNOCELLULOSIC SUGAR
WITH WOOD-DERIVED ORGANIC ACIDS, INCLUDING CO-CULTURE
This example employs strains selected for preferred growth using organic acid
under
heterotrophic or mixotrophic conditions for two types of products, pigments
and recombinant
nucleic acids such as dsRNA or recombinant protein products. It is
exemplified, but not
limited to, using Haematococcus pluvialis and Chlamydomonas reinhardtii in
dark
cultivation, used alone as monocultures or in combination as co-cultures; as
well as using
Haematococcus pluvialis with a second cell type other than Chlamydomonas; the
latter is
exemplified but not limited to Scenedesmus obliquus, KAS1003, a Hawaiian
accession
previously confirmed by DNA fingerprinting as described (Kuehnle et at. 2015).
Southern pine lignocellulosic hydrolysate is overlimed and then further pH-
adjusted
with sulfuric acid to pH 5 prior to use for bacterial fermentation for
bioconversion of sugars
to acetic acid as known in the art (Mohagheghi et at. 2006), for example using
Moorella
thermoacetica ATCC 39073 (Clostridium thermoaceticum) according to Ehsanipour
et at.
(2016). The resultant solution, sustained at pH 6.8, contains about 1%
unconverted
lignocellulosic simplified sugars (glucose and minor C6 sugars) in the
presence of 2% wood-
derived acetic acid. For the purposes of algal culture, a portion of the 2%
wood-derived acetic
acid/1% wood-derived glucose is diluted 33.3x to 0.06% acetic acid (10 mM
acetic acid) and
0.03% glucose (1.65 mM glucose) in growth medium (F with nitrate replaced with
equal
molar urea and 1/10th yeast extract by weight of total carbon sources
present), pH adjusted to
7 and filter-sterilized by 0.2-micron cross flow filtration to supply the
initial acetic acid to
start the fermentation. Also a portion of the 2% wood-derived acetic acid is
concentrated 5x
such as by distillation as known in the art to 10% acetic acid/5% glucose (or
greater), pH 4, to
supply carbon throughout the algae fermentation run. It is understood that
higher acetic acid
concentrations or purified slipstreams allow smaller volume increases in the
fermentation
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tank and is preferred for very high cell density cultures. Alternatives
include use of a
multistep process to generate acetic acid at desired concentrations, such as
with Acetobacter
and prior ethanol conversion by Saccharomyces as is known in the art, as is
using other
efficient mutants of hom*oacetogens for a one-step process. Fungal species in
addition to
bacterial species are known in the art to produce high amounts of organic
acids; notable
genera include Aspergillus and Rhizopus.
The microalgal species are Haematococcus pluvialis KAS1601 (an improved strain
of
H. pluvialis UTEX 2505, Culture Collection of Algae at the University of Texas
Austin, Tex.,
USA) and Chlamydomonas reinhardtii KAS1001 (137C, Chlamydomonas Resource
Center,
St. Paul, Minn. USA). These are maintained heterotrophically in 0.06% wood-
derived acetic
acid/0.03% wood-derived glucose medium and then transferred to preferred
growth media for
heterotrophic culture on acetic acid, using media as described in US Serial
No. 62/356,896,
with sodium acetate replaced with 0.06% wood-derived acetic acid/0.03% wood-
derived
glucose, adjusted to pH 7. The fermentation uses a 2.3 L fermentation vessel
(New
Brunswick BioFlo 115) at 1 L operated using BioCommand software with
peristaltic pumps,
and head plate ports. The pH is maintained at pH 7.7 to 7.3 for the duration
of the fed-batch
fermentation with pH-triggered additions of the concentrated 5x to 10% acetic
acid/5%
glucose (or greater), pH 4, and other inputs are monitored and maintained as
described in US
Serial No. 62/356,896. Carbon (10% wood-derived acetic acid/5% wood-derived
glucose) is
supplied throughout the fermentation run from 75 IAL/L per hour up to 1500
L/L per hour or
more as the culture density increases.
The sole sources of fixed carbon inputs are the unconverted lignocellulosic
simplified
sugars and the bioconverted wood-derived acetic acid. For Chlamydomonas
reinhardtii
KAS1001 cultivated as a monoculture in fermentation over 120 hours, an initial
0.4 g/L algal
culture produces a biomass with density of 6.5 g/L (specific growth rate
0.57/d). This is the
first instance of biomass of this genus being cultivated in wood-derived
feedstocks.
Subsequently a C. reinhardtii KAS1402 is plastid-transformed as known in the
art to carry
an inverted repeat for a mosquito 3-HKT gene fragment per US Serial No.
62/356,896. A
selected KAS1402 event that carries the 3-EIKT dsRNA coding sequence when
cultured
under heterotrophic conditions on acetic acid or in combination with lactic
acid reaches cell
densities of 30, 50, and 85 g/L. A BioFlo 610 model 120 L vessel containing 90
L of media
is fed nutrients as required for growth via automated feeding of nutrient
concentrates; carbon
feed and the pH of the culture is maintained between 6.9 and 7.6 using a 20%
acetic acid
concentrate. Oxygen is supplied by agitation at 500 rpm with 100 1pm gas flow
with pure
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oxygen supplying up to 50% of the total gas flow. It is also shown that the
Chlamydomonas
grows on the wood-derived organic acid stream alone, in the absence of
simplified sugars, to
similar yields. This productivity greatly surpasses what is known in the art
for using acetate
including: ammonium acetate, sodium acetate, or potassium acetate.
For H. pluvial's, cells redden as ammonium concentration increases (2.5 mM and
above). To mitigate the ammonium build-up during biomass generation,
Scenedesmus
obliquus KAS1003 is co-cultured at low density with the H. pluvialis KAS1601,
as described
in US Serial No. 62/356,896. The initial C6 sugars (0.3 g/L) needed by KAS1003
are
supplied initially by the 33.3x dilution of 2% wood-derived acetic acid/1%
wood-derived
glucose as described above. Carbon (10% wood-derived acetic acid/5% wood-
derived
glucose) is supplied throughout the fermentation run from 75 L/L per hour up
to 1500 L/L
per hour or more as the culture density increases to supply both acetic acid
and glucose. The
initial ratio of KAS1003 to KAS1601 is such that KAS1601 produces more
ammonium than
the KAS1003 can consume so the ammonium concentration reaches >2.5 mM by 96
hours of
fermentation (or, glucose and nutrients except urea and phosphate can stop
being fed at 72
hours which allows the ammonium to reach >2.5 mM by 96 hours). The culture is
allowed to
ferment for an additional 24 hours to increase the astaxanthin content of the
motile KAS1601
cells. For Haematococcus KAS1601 in unoptimized fermentation over 120 hours,
an initial
0.2 g/L algal culture produces a biomass with density of 3 g/L, 1.2% to 2%
pigment and 45%
to 50% protein content. Base, unoptimized, usage of feedstock is about 4.2 mL
glacial acetic
acid equivalents required for every gram algal biomass using H. pluvial's. A
preferred
compositional profile for use of the intact biomass for aquaculture feed is
possible by
selecting the finishing step of urea or sulfate stress to obtain corresponding
protein and
pigment content desired by feed formulators and end users. A KAS1601 and
KAS1003 (S.
obliquus) fermentation with an initial cell density of 2 g/L produces 32 g/L
biomass in 120
hours; an initial 3 g/L produces 48 g/L in 120 hours with 1.2% pigment and 45%
protein with
vegetative culture under sulfate stress. Agitation is with a pitched blade
impeller at 350 rpm
with gas flow at 1 vessel volume per minute and pure oxygen supplied as needed
to maintain
dissolved oxygen at > 50%.
It is understood that the process can be optimized for each cell type and to
select a
preferred duration of the production cycle while achieving product formation,
and to select a
preferred compositional profile for target market use. In this example, it is
understood that
the S. obliquus can be interchanged with a different microbial cell type
suited to heterotrophic
growth as long as it still prefers a fixed carbon source that is not an
organic acid,
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preferentially glucose or xylose, and preferentially consumes ammonium as
nitrogen source,
as is known in the art for many such cell types. Options among astaxanthin or
other pigment
producing cell types, or for oil-producing or high beta-glucan-producing cell
types, are other
species of Scenedesmus, Chlorella, Auxenochlorella, Monoraphidium, Euglena,
Rhodotorula,
and many different diatoms such as Phaeodac0;lum and Cyclotella, and
thraustochytrids or
thraustochytrid-like cell types, as known in the art.
Alternatively, ammonium control proceeds using H. pluvialis co-culture with
Chlamydomonas reinhardtii, per US Serial No. 62/356,896. The final biomass is
comprised
of about 99% H. pluvialis biomass, similar to what may occur naturally in an
open pond with
mixed microorganisms. Adjustment of co-cultivation parameters such as dosing
of the cell
types allows reaching different target rates of growth and productivity
relative to the
carotenogenesis trigger for H. pluvialis of about 2.5 mM ammonium.
It is understood that the process can also be optimized for the composition of
the
hydrolysate that is produced, depending on the hydrolysis process and type of
wood
processed by any particular mill, and the degree of dilution/concentration of
the hydrolysate.
Some compositions and profiles are known in the art, examples of which are
described by
Burkhardt et at. (2013); Brodeur et at. (2011); Harmsen et at. (2010); and
Chaturvedi et at.
(2013). Concentrated hydrolysate is optionally prepared from desalted or
otherwise
"conditioned" solution derived from hydrolysis of pretreated material that was
washed to
remove extractives, using methods known in the art known and described in
US20100151538
and US20110318798.
EXAMPLE 6. PRODUCTION OF RECOMBINANT ALGAL BIOMASS IN WOOD-
DERIVED LIGNOCELLULO SIC FEED STOCK
This non-limiting example is directed to a recombinant algal cell that is
cultured by
the methods of the invention. In one embodiment, Chlamydomonas is cultured
with a
preferred culture medium comprising a wood-derived organic acid and a wood-
derived
lignocellulosic simplified sugar. A second case provides for a different
recombinant algal
cell of Scenedesmus that is cultured with a preferred culture medium
comprising wood-
derived lignocellulosic simplified sugar in the presence of a process residual
of wood
lignocellulose hydrolysis.
Heterotrophically adapted transgenic algae are maintained in 250 mL volumes in
500
mL flasks on an orbital shaker at 100 rpm at 28 C, initial pH of 7.0 as for
Example 5 except
urea is replaced with NH4C1 for KAS1003. Both species of transgenic algae
carry pChlamy 2
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that contains the Aph7 (hygromycin resistance) gene under control of beta-
tubulin promoter
in their nucleus. Inoculum for the fermentor uses cells that are pelleted and
re-suspended
in wood-derived concentrates standardized to hydrocarbon. Fermentation
proceeds as
described in Example 5 above, using the reactor conditions disclosed in
Example 3 of US
Serial No. 62/356,896 except pure carbons sources are replaced with wood
hydrolysates.
Also the pH in KAS1003 fermentation is maintained at 7.4 with 0.5 M NaOH.
Elucidation of
transgene expression measures gene transcription by qRT-PCR. Primers designed
using
Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) monitor endogenous actin
gene
transcription compared to that of the transgene. Primers for actin in KAS1003
amplify 187
bp (5' -GATGCCCTGAGGTACTGTTC-3' (SEQ ID NO: 1) and
5'-
ACCTCCTTGCTCACTCTGTC-3' (SEQ ID NO: 2)) and in KAS1001 amplify 163 bp (5'-
ATAGCTTGCGTCTGGATAGG-3' (SEQ ID NO: 3) and 5'-
TGCGTCCTCTCATGTAAAAA-3' (SEQ ID NO: 4)). Primers for Aph7 amplify a 111 bp
(5'- CAACATCTTCGTGGACCTG-3" (SEQ ID NO: 5) and 5'-
AAGGCGTTGAGATGCAGT-3' (SEQ ID NO: 6)). RNA is extracted from freeze dried
biomass after 72 hr culture from dark-grown biomass at log phase (72 hrs)
following
manufacturer's instructions for RNeasy Plant Mini Kit (Qiagen 74903) and qRT-
PCR
performed following manufacturer's instructions for Superscript III One Step
RT-PCR kit
(Invitrogen 12574-018). The resulting qRT-PCR products quantification is
compared relative
to the non-transgenic control; KAS1003 has Aph7 expression 1.3 fold higher
than actin and
KAS1001 has Aph7 1.5 fold higher than actin. It is understood that a gene of
interest can be
further employed in transgenic algae as is known in the art using this
expression system. For
a target recombinant molecule at this volume of production at a mill-based
biorefinery, these
may be expressed compounds that confer animal or fish health as part of a
whole biomass
addition to the feed formulation, and further, may include those that
accumulate the highest
during the rapid biomass growth stage.
EXAMPLE 7¨ ADDITIONAL HETEROTROPHIC CO-CULTIVATION OF SPECIES IN
WOOD-DERIVED LIGNOCELLULO SIC FEEDSTOCK
This example illustrates the potential of full utilization of sugars present
in wood
hydrolysates using a co-culture. Chlorella KAS908 and Rhodotorula KAS1101 are
individually grown at 7-1_, fermentor scale in a medium containing model
sugars to evaluate
biomass growth and sugar utilization patterns on the major fixed carbons in
Southern
Hardwood Chips. With the previously determined fermentation conditions (see
Example 1),
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KAS908 is grown in 2F+YE medium containing glucose and xylose at 19.43 g/L and
8.06
git, respectively. These sugar concentrations mimicked the sugar composition
of Southern
Hardwood Chips hydrolysate. KAS1101 is grown in F/2 +YE with the same glucose
and
xylose concentrations and fermentation conditions. Both strains show an
increase in biomass
productivity at different rates. KAS908 has a biomass productivity of 1,24
g/L/day and
KAS1101 has a biomass productivity of 1.5 (A/day. KAS908 and KAS1101 differ in
the
rates of glucose utilization, with the yeast depleting the glucose much more
rapidly (Figure
7). This suggested that, if starting with the same culture density in a co-
culture, the yeast
may outcompete the chlorophyte over time.
Dark shake flask experiments (heterotrophic) demonstrate the feasibility of
biomass
production by co-cultures of KAS908 (initial 0D750 = 0.2) and KAS1101 (initial
0D750 =
0.2) on culture media with xylose/arabinose C5 sugars and glucose C6 sugar, or
those sugars
plus process residuals in a wood hydrolysate. Treatments are as follows, using
cell cultures
that are previously acclimated under heterotrophic conditions: a) F+YE+
glucose (9 g/L); b)
F+YE+ glucose (4 g/L) + arabinose (2.5 g/L) + xylose (2.5 g/L); c) F+YE +
arabinose (4.5
g/L) + xylose (4.5 g/L); and d) 30% SHC hydrolysate solution (equivalent to 9
g/L glucose)
with F + YE.
All media treatments support growth of the co-cultures over a 6-day period.
The co-
culture on glucose alone stays green through day 6. The cultures grown in the
media
containing the glucose-arabinose-xylose blend or grown in the SHC hydrolysate
are an
orange-green by day 6, indicating the faster growth of the red yeast and its
ability to utilize
both C6 and C5 sugars present in the wood hydrolysate for growth. The co-
culture grown in
C5 arabinose-xylose sugars alone produces an eventual change to reddish-brown
color
similar to a KAS1101 monoculture, illustrating a faster growth of the
Rhodotorula over the
Chlorella on this substrate. To confirm that KAS1101 utilizes C5 sugars alone,
a parallel
growth experiment is carried out using F/2 medium containing only the C5
sugars: a)
F/2+YE+ arabinose (9 g/L); and b) F/2+YE +Xylose (9 g/L). Visual appearance of
the flasks,
with their more opaque cultures compared to the starting cultures, indicates
that KAS1101
indeed utilizes C5 sugars (i.e., arabinose and xylose) for growth.
EXAMPLE 8 ¨ ASSESSMENT OF ADDITIONAL SPECIES PERFORMANCE IN
WOOD-DERIVED LIGNOCELLULO SIC FEEDSTOCK
Several algal species of commercial interest (Chlorella zofingiensis,
Parachlorella
spp., Scenedesmus obhquus and Mayamaea spp.) are tested for growth, pigments,
and lipids
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on different wood hydrolysates at flask bioreactor level. This includes
comparison with the
model sugars and comparison of the different hydrolysate sources: Southern
Hardwood
Chips (SHC, 3% total sugar), Southern Pine Bleached Kraft (SPBK, 9% total
sugar),
Hardwood Chips (HWD, 4.4% total sugar) and Southern Pine Finer chips (SPFC,
2.15% total
sugar). Chlorella zofingiensis KAS1170 (UTEX32) is grown in SHC, SPFC and HWD
(normalized to 2 g/L total sugars C6 + C5). Hawaiian Parachlorella KAS741 is
grown in
HWD (normalized to 2 g/L total sugars). Briefly, the wood hydrolysate solution
is
supplemented with F medium components and adjusted to pH 7.0 as per Example 1.
Heterotrophically adapted KAS1170 is grown in F (as in Example 1) to log phase
mixotrophically and photosynthetically on a 16/8 (day/night) cycle (i.e., to
allow full
depletion of residual glucose in the cultures before inoculating into wood
hydrolysate-
containing medium). To F medium containing the normalized 2 g/L sugar is added
0.2 g/L of
yeast extract. To F medium containing wood hydrolysate normalized to 4 g/L is
added 0.4
g/L yeast extract. All media are inoculated to an initial density of 1.0'6
cells/mL. Cultures in
shake flasks are allowed to grow for 7 days in the dark on an orbital shaker
(INNOVA 4000
incubator shaker) at 28 C and 120 rpm. Growth of KAS1170 (as dry weight) as
well as
glucose utilized are compared. Growth (DW) and pigment profile of KAS741 in
HWD and
model sugars are also compared. Biomass obtained is analyzed by TLC for
pigment profiles
using hexane:acetone (3:1) as running buffer on TLC plates (silica gel matrix,
Sigma
#Z122777)
Across the board, growth was observed in all wood hydrolysates using Chlorella
zofingiensis and Parachlorella spp., with better growth from SPFC and HWD
compared to
model sugars. Among all the wood hydrolysates tested, KAS1170 grown in the
softwood
Southern Pine SPFC showed the highest biomass production with a 600-fold
increase
compared to the model sugars having a 400-fold increase (from Day 0) and a
corresponding
glucose utilization to biomass ratio of 1:0.81 (w/w) compared to 1: 2.4 for
the control on
model sugars, indicating unexpected contributions to growth from the process
residuals. Both
KAS1170 and KAS741 grown on HWD also showed higher increase in biomass
production
than the model sugars, by 500-fold compared to less than 100-fold for KAS1170,
and by 300-
fold compared to 150-fold for KAS741 (from Day 0). KAS741 culture shows
notable
increase in viscosity from exopolysaccharide production, demonstrating that
wood
hydrolysates are suited to producing this phenotype and product. The
exopolysaccharide can
be separated from the cells and dried into a mass. Although KAS1170 had an
increase in
growth on SHC (88%) and SPBK (64%), there was a higher increase in biomass
production
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on the model sugars of 1650% and 125%, respectively. KAS741 grown in hardwood
HWD
hydrolysate shows much lower pigmentation than on model sugars. KAS1170 shows
lutein
and astaxanthin contents varying among the different wood hydrolysates.
KAS1170 grown
in SHC hardwood hydrolysate has higher astaxanthin and lower lutein than model
sugars.
KAS1170 on softwood SPFC shows higher astaxanthin and lutein content than
model sugars.
KAS1170 on softwood SPBK and equivalent model sugars without process residuals
have
similar astaxanthin and lutein contents.
Scenedesmus obliquus KAS1003 and the diatom Mayamaea spp KAS1111 (See
Kuehnle et at. 2015 for identification by DNA fingerprinting) are grown at 22
C at 100 rpm
in the dark in modified F medium containing 1.28 g/L glucose and 0.72 g/L
xylose with
nitrate replaced with equal molar NH4C1 as the nitrogen source. A 25 mL of the
log phase
culture is used to inoculate 225 mL of control medium (same as previous) or to
inoculate 225
mL (26.75 mL of hydrolysate labeled "Hardwood", HWD, and 198.25 mL growth
medium,
pH adjusted to 7.0 with 1M Tris-HC1). Both the control medium and hydrolysate
medium
contained 1.28 g/L glucose and 0.72 g/L xylose at the start of the
fermentation. On the third
day a 100 mL sample was taken for analysis; the biomass was spun down at 3000
rpm for 5
minutes and freeze dried and the supernatant was collected for glucose
analysis. On the fifth
day another 100 mL of the remaining culture was collected for the same
analysis performed
on the third day of fermentation. Biomass
was analyzed for pigments (TLC,
spectrophotometer) and phospholipids (TLC). Total pigments and PLs were
extracted from
freeze dried ground biomass using 50 [.tt of chloroform:methanol (2:1) per mg
of biomass.
Debris was cleared by centrifugation at 8,000 rpm for 5 minutes. For pigments,
absorbance
at 470 nm was used to estimate differences in pigment content of crude
extracts. For
phospholipids, the 0.5 mg equivalent biomass was loaded onto a silica matrix
gel (Sigma
#Z233888) and were separated using chloroform:methanol:water (65:25:4) as the
running
buffer. Phospholipids were stained with molybdenum Blue spray reagent (Sigma
M1942)
and observed 20 minutes thereafter.
Results for KAS1003 showed 40% more biomass by day 3 (log phase) with glucose
running out between day 3 and day 5. By day 5 (stationary phase, low glucose)
the amount
of biomass in each culture was equal but the hardwood HWD sample contained
1.5x more
pigments than the control. Pigments from 0.2 mg equivalent biomass were
separated by TLC
run on silica gel matrix (Sigma #Z122777) using hexane:acetone (3:1) as
running buffer, the
bands for beta-carotene and lutein/zeaxanthin were observed in all samples, no
detectable
astaxanthin in any samples. We have demonstrated elsewhere that KAS1003 will
generate
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astaxanthin in fermentation cultures once sufficient nutrient (N) deficiency
occurs, and that
did not occur in these 5 day old cultures in the treatment or controls. Once
the specific growth
rate is optimized for the KAS1003 using the preferred wood hydrolysate for a
select pulp and
paper mill, it is known in the art to time nitrogen feed during fed-batch
fermentation for
sufficient depletion to reach favorable specific productivity for a pigment
such as astaxanthin.
For lipids, measured as phospholipids content during growth phase before
glucose depletion,
there was no observable change on day 3 in the HWD and the control samples.
The diatom Mayamaea spp KAS1111 was grown and harvested in the same manner
as above for KAS1003. Total pigments and phospholipids were extracted from
freeze dried
ground biomass using 50 uL of chloroform:methanol (2:1) per mg of biomass.
Debris was
cleared by centrifugation at 8,000 rpm for 5 minutes. Fucoxanthin from 0.2 mg
of biomass
was separated by TLC run on silica gel matrix (Sigma #Z122777) using
hexane:acetone (3:1)
as running buffer, the fucoxanthin band was cut out and eluted in acetone for
absorbance at
470 nm readings. Fucoxanthin content was estimated on a dry weight basis by
comparing to a
dilution gradient of absorbance at 470 nm of commercially available
fucoxanthin (Sigma
F6932). Biomass generated was equal for both control and hardwood HWD samples
for both
day 3 and day 5. On day 3 (log phase) the control (0.55% pigment per DW) had
5x more
fucoxanthin than the HWD (0.11% DW) sample; by day 5 (stationary phase, in
which silica
is lacking and glucose and ammonium were present) the control (0.63% pigment
per DW)
had 7x more fucoxanthin than the HWD (0.09% DW) sample. Unlike
other
carotenoids/xanthophylls, fucoxanthin is not a stress-induced pigment.
Phospholipids content,
monitored as a measure of lipids during the growth phase before glucose
depletion, was
similar on day 3 in the HWD and the control samples under the test conditions
used.
Taken together, these results indicate the following advantages conferred
using the
method of the invention: Rapidly increased biomass yield, or reduced
fermentation cycle
time, using diluted hardwood and softwood hydrolysates; larger cells in some
cases using
hardwood hydrolysate when under higher osmotic pressure, useful for downstream
processing; increased pigment yield in some hydrolysates for commercially
valued pigments;
and a strategy to use the method of the invention with added stress to
generate additional
desired pigment (astaxanthin)
Further, using a diatom microalga with the method of the invention, the
hardwood
hydrolysate is seen to support growth (biomass production) very similar to the
control
medium lacking the lignocellulosic hydrolysis process residuals, and to
significantly decrease
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the pigmentation of the biomass. This is advantageous for use of biomass in
products where
added color is unwanted.
EXAMPLE 9- MIXOTROPHIC CULTIVATION AND PRODUCT FORMATION USING
WOOD-DERIVED LIGNOCELLULOSIC HYDROLYSATES
While these examples are largely exemplified for dark heterotrophic
cultivation, it is
understood that the methods of the invention can be applied without limitation
to mixotrophic
cultivation applicable for those species that are not obligate heterotrophs
and are facultative
heterotrophs. As a result of mixotrophy, productivity increases. This example
is a
modification of Example 5, such that heterotrophic growth of Haematococcus
pluvialis
KAS1601 is replaced by mixotrophic growth under 30 [tE light. The growth rate
of H.
pluvialis KAS1601 is accelerated over dark culture such that a culture
starting with 0.05 g/L
biomass reaches an initial 9.0 g/L in 96 hours, with a pigment content of 2.4%
DW. It is
understood that higher productivities are obtainable with adjustment in
feedstock and
nutrition following mass balances, seed densities, aeration, and mixing, and
light delivery, as
is known in the art. When not used as whole-cell feed, pigment-extracted
biomass as is
known in the art is also suited as meal for fish, insect and animal feed
applications, with the
protein, beta-glucan, vitamins, micronutrients and residual pigment providing
growth and
health benefits.
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