MICROBIAL CONSORTIA
CROSS REFERENCE TO RELATED APPLICATIONS
This claims the benefit of U.S. Provisional Application No. 62/1 26,343, filed February
27, 2015, which is incorporated herein by reference in its entirety.
FIELD
This disclosure relates to microbial consortia and methods of use of the microbes
included in the consortia, particularly for biodegradation and agricultural processes and uses.
BACKGROUND
World food demand continues to increase under pressure of increasing population
growth. However, agricultural workers are faced with shrinking amounts of land available for
agriculture, soil depletion, and changing environmental conditions, among other challenges.
Thus, there is a need to develop compositions and techniques that can increase food production,
while also decreasing the use of potentially harmful herbicides, insecticides, and fungicides.
SUMMARY
Disclosed herein are microbial consortia and compositions including microbes for use in
agricultural or biodegradation applications. In some embodiments, a microbial composition of
the present disclosure is the microbial consortium deposited with the American Type Culture
Collection (ATCC, Manassas, VA) on November 25, 2014 and assigned deposit number PTA-
121755 (referred to herein as A1006) or a composition including some or all of the microbes in
A1006. In other embodiments, a composition of the present disclosure includes microbes from
five or more microbial species selected from Bacillus spp., Pseudomonas spp., Lactobacillus
spp., Desulfococcus spp., Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp.,
Ruminococcus spp., Leptospirillum spp., Halorhabdus spp., Clostridium spp., Xenococcus spp.,
Cytophaga spp., and Candidates spp. In some embodiments, the composition further includes
one or more of Microbacterium spp., Sporosarcina spp., Lysinibacillus spp., Nesterenkonia spp.,
Agrococcus spp. , and Acremonium spp. In additional embodiments, the composition includes
microbes from five or more (such as 5, 10, 15, 20, 25, or more) of the microbes listed in Table 1.
The disclosed compositions may also include additional components, including but not limited
to one or more of additional microbe species, chitin, chitosan, glucosamine, and/or amino acids.
Also disclosed are agricultural uses of the disclosed microbial consortia or compositions.
In some embodiments, the methods (uses) include contacting soil, plants, and/or plant parts
(such as seeds, seedlings, shoots, leaves, stems, or branches) with a disclosed microbial
consortium (such as A1006), a composition including some or all of the microbes from A1006,
or a composition including five or more of the microbial species listed in Table 1. The
microbial consortia or microbe-containing compositions may be applied to soil, plant, and/or
plant parts alone or in combination with additional components (such as additional microbes,
chitin, chitosan, glucosamine, amino acids, and/or soil supplements or fertilizer, such as liquid
fertilizer).
In additional embodiments, the disclosed microbial consortia or compositions including
microbes are used in methods of degrading biological materials, such as chitin-containing
biological materials. In some examples, the chitin-containing materials are mixed with a
microbial consortium (such as A1006) or a composition including five or more of the microbial
species listed in Table 1 and fermented to produce a fermented mixture. The fermented mixture
optionally may be separated into solid and liquid fractions. The fermented mixture or fractions
produced therefrom can be used in agricultural applications in combination with the disclosed
microbial consortia or compositions or can be used in further degradation processes, for example
to produce increased levels of degradation products in the fractions.
The foregoing and other features of the disclosure will become more apparent from the
following detailed description, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic showing an exemplary fermentation process used to obtain the
A1006 microbial consortium.
FIG. 2 is a schematic showing an exemplary process for biodegradation of a chitincontaining
biological material (exemplified as shrimp waste) with a disclosed microbial
consortium or microbial composition.
FIG. 3 is a schematic showing an exemplary process for biodegradation of chitin with a
disclosed microbial consortium or microbial composition (such as A1006).
FIGS. 4A-4C are graphs showing growth curves of Al 006 exposed to liquid ureaammonium
nitrate (UAN 32) fertilizer or control culture at 4°C (FIG. 4A) or 23°C (FIG. 4B)
and then cultured under aerobic or anaerobic conditions. FIG. 4C includes data from FIG. 4B,
plus growth curve of A1006 exposed to 10-34-0 ammonium phosphate (AP) liquid fertilizer at
23°C prior to aerobic or anaerobic culture.
FIGS. 5A-5G are graphs showing the effect on yield of treatment of corn with a
microbial composition (FIGS. 5A-5C and 5E), HYTb (FIGS. 5D and 5F), or a microbial
composition under water stress conditions (FIG. 5G).
FIGS. 6A-6D show the effect on yield of treatment of wheat with a microbial
composition (FIGS. 6A-6B) or with a microbial composition plus HYTb (FIG. 6C). FIG. 6D is
a digital image showing roots of wheat plants treated with a microbial composition plus HYTb
(test) compared to control plants.
FIGS.7A-7E are a series of graphs showing the effect on yield of treatment of tomato
with A1006 (five trials, FIGS. 7A-7E, respectively).
FIG. 8 is a graph showing the effect on yield of treatment of sunflower with a microbial
composition.
FIG. 9 is a graph showing the effect on yield of treatment of rice with a microbial
composition.
FIGS. 10A-10B show the effect on yield of treatment of soybean with a microbial
composition (FIG. 10A) or with a microbial composition plus HYTb (FIG. 10B).
FIG. 11 is a graph showing the effect on yield of treatment of strawberry with a
microbial composition plus HYTb.
FIG. 12 is a graph showing the effect on yield of treatment of beetroot with a microbial
composition plus HYTb.
FIGS. 13A and 13B are graphs showing the effect on yield of treatment of green cabbage
with a microbial composition plus HYTb in two trials (FIGS. 13A and 13B, respectively).
FIG. 14 is a graph of a cucumber vigor assay showing first leaf area index (LAI) on day
18 in plants treated with HYTa (Al 006). *p<0.01 by ANOVA analysis.
SEQUENCE LISTING
Any nucleic acid and amino acid sequences listed herein or in the accompanying
sequence listing are shown using standard letter abbreviations for nucleotide bases and amino
acids, as defined in 37 C.F.R. § 1.822. In at least some cases, only one strand of each nucleic
acid sequence is shown, but the complementary strand is understood as included by any
reference to the displayed strand.
SEQ ID NOs: 1 and 2 are forward and reverse primers, respectively, used to amplify 16S
rDNA from Al 006.
DETAILED DESCRIPTION
In nature, the balance of microbial species in the soil is influenced by soil type, soil
fertility, moisture, competing microbes, and plants (Lakshmanan et al. , Plant Physiol. 166:689-
700 2014). The interplay between microbial species and plants is further affected by agricultural
practices, which can improve or degrade the soil microbiome (Adair et al., Environ. Microbiol.
Rep. 5:404-413 2013; Carbonetto et al,PLoS One 9:e99949 2014; Ikeda et al., Microbes
Environ. 29:50-59 2014). Fertile or highly productive soils contain a different composition of
native microbes than soil that is depleted of nutrients and linked to low crop productivity.
Different microbial species are associated closely with plants, on the above ground plant
surfaces in the phyllosphere, at the root surface in the soil rhizosphere, or intimately as
endophytes. Large-scale DNA analysis of these microbe associations has revealed unexpected
phylogenetic complexity (Rincon-Florez et al., Diversity 5:581-612 2013; Lakshmanan et al,
Plant Physiol. 166:689-700 2014). Studies have determined complex microbiomes can be
correlated to plant productivity, crop yield, stress tolerance, secondary metabolite accumulation,
and disease tolerance (Bhardwaj et al., Microbial Cell Factories 13:66-75, 2014; Vacheron et
al., Frontiers Plant Science 4:1-19 2014). Furthermore, plants can specifically select the
microbial mixtures from the local environment and potentially fine-tune the microbiome at the
level of crop variety (Hartmann et al., Plant Soil 321:235-257 2009; Doornbos et al.,Agron.
Sustain. Dev. 32:227-243 2012; Marasco et al, PLoS One 7:e48479 2012; Peiffer et al., Proc.
Natl. Acad. Sci. USA 110:6548-6553; Bulgarelli et al, Ann. Rev. Plant Biol 64:807-838 2014).
Root-associated microbes can promote plant and root growth by promoting nutrient
cycling and acquisition, by direct phytostimulation, by mediating biofertilization, or by offering
growth advantage through biocontrol of pathogens. Agriculturally useful populations include
plant growth promoting rhizobacteria (PGPR), pathogen-suppressive bacteria, mycorrhizae,
nitrogen-fixing cyanobacteria, stress tolerance endophytes, plus microbes with a range of
biodegradative capabilities. Microbes involved in nitrogen cycling include the nitrogen-fixing
Azotobacter and Bradyrhizobium genera, nitrogen-fixing cyanobacteria, ammonia-oxidizing
bacteria {e.g., the genera Nitrosomonas and Nitrospira), nitrite-oxidizing genera such as
Nitrospira and Nitrobacter, and heterotropWc-denitrifying bacteria (e.g, Pseudomonas and
Azospirillum genera; Isobe and Ohte, Microbes Environ. 29:4-16 2014). Bacteria reported to be
active in solubilization and increasing plant access to phosphorus include the Pseudomonas,
Bacillus, Micrococcus, and Flavobacterium, plus a number of fungal genera (Pindi et al, J.
Biofertil Biopest. 3:4 2012), while Bacillus and Clostridium species help solubilize and
mobilize potassium (Mohammadi et al, J. Agric. Biol. Sci. 7:307-316 2012). Phytostimulation
of plant growth and relief of biotic and abiotic stresses is delivered by numerous bacterial and
fungal associations, directly through the production of stimulatory secondary metabolites or
indirectly by triggering low-level plant defense responses (Gaiero et al, Amer. J. Bot. 100:1738-
1750 2013; Bhardwaj et al, Microbial Cell Factories 13:66-76 2014).
In addition to activity in the environment, microbes can also deliver unique
biodegradative properties in vitro, under conditions of directed fermentation. Use of specific
microbial mixtures to degrade chitin and total protein can yield new bioactive molecules such as
free L-amino acids, L-peptides, chitin, and chitosan known to enhance growth or boost stress
tolerance via activation of plant innate immunity (Hill et al, PLoS One 6:e 19220 201 1; Tanaka
et al, Plant Signal Behav. E22598-147 201 3). Specific microbial communities can serve
multiple tasks, by delivering unique fermentation breakdown products, which are themselves
biologically beneficial to crops, plus the resultant microbial consortium, which can be delivered
as an agricultural product to enhance crop productivity.
As described herein, consortia of aerobic and/or anaerobic microbes derived from fertile
soil and marine sources have been successfully co-fermented and stabilized, offering direct
growth and yield benefits to crops. Enzymatic activity of these microbial mixtures has further
yielded fermentation products with chitin, glucosamine, protein, and/or amino acids. In some
embodiments, direct delivery of microbial consortia and/or compositions can allow early root
colonization and promote rhizosphere or endophytic associations. In some embodiments,
benefits of delivery of microbial consortia to plants include one or more of increased root
growth, increase root hair production, increased root surface area, stronger plants able to
withstand transplantation shock, faster stand establishment, resistance to abiotic stress, and
higher plant productivity and yield. Complex microbial mixes can span across plant species and
genotypes, interacting with microbial soil communities to offer benefits to a wide range of crops
growing under different agricultural conditions.
I. Terms
Unless otherwise noted, technical terms are used according to conventional usage.
Definitions of common terms in molecular biology may be found in Krebs et al, Lewin 's Genes
XI, published by Jones and Bartlett Learning, 2012 (ISBN 1449659853); Kendrew et al. (eds.),
The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN
0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by Wiley, John & Sons, Inc., 2011 (ISBN 8126531789); and George
P. Redei, Encyclopedic Dictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003
(ISBN: 0-471-26821-6).
The following explanations of terms and methods are provided to better describe the
present disclosure and to guide those of ordinary skill in the art to practice the present
disclosure. The singular forms "a," "an," and "the" refer to one or more than one, unless the
context clearly dictates otherwise. For example, the term "comprising a cell" includes single or
plural cells and is considered equivalent to the phrase "comprising at least one cell." As used
herein, "comprises" means "includes." Thus, "comprising A or B," means "including A, B, or A
and B," without excluding additional elements. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference in their entirety for all
purposes. In case of conflict, the present specification, including explanations of terms, will
control.
Although methods and materials similar or equivalent to those described herein can be
used to practice or test the disclosed technology, suitable methods and materials are described
below. The materials, methods, and examples are illustrative only and not intended to be
limiting.
To facilitate review of the various embodiments of this disclosure, the following
explanations of specific terms are provided:
Aquatic Animal: An animal that lives in salt or fresh water. In particular embodiments
disclosed herein, an aquatic animal includes aquatic arthropods, such as shrimp, krill, copepods,
barnacles, crab, lobsters, and crayfish. In other embodiments, an aquatic animal includes fish.
An aquatic animal by-product includes any part of an aquatic animal, particularly parts
resulting from commercial processing of an aquatic animal. Thus, in some examples, aquatic
animal by-products include one or more of shrimp cephalothorax or exoskeleton, crab or lobster
exoskeleton, or fish skin or scales.
Contacting: Placement in direct physical association, including both in solid and liquid
form. For example, contacting can occur with one or more microbes (such as the microbes in a
microbial consortium) and a biological sample in solution. Contacting can also occur with one
or more microbes (such as the microbes in a microbial consortium) and soil, plants, and/or plant
parts (such as foliage, stem, seedling, roots, and/or seeds).
Culturing: Intentional growth of one or more organisms or cells in the presence of
assimilable sources of carbon, nitrogen and mineral salts. In an example, such growth can take
place in a solid or semi-solid nutritive medium, or in a liquid medium in which the nutrients are
dissolved or suspended. In a further example, the culturing may take place on a surface or by
submerged culture. The nutritive medium can be composed of complex nutrients or can be
chemically defined.
Fermenting: A process that results in the breakdown of complex organic compounds
into simpler compounds, for example by microbial cells (such as bacteria and/or fungi). The
fermentation process may occur under aerobic conditions, anaerobic conditions, or both (for
example, in a large volume where some portions are aerobic and other portions are anaerobic).
In some non-limiting embodiments, fermenting includes the enzymatic and/or non-enzymatic
breakdown of compounds present in aquatic animals or animal by-products, such as chitin.
Liquid fertilizer: An aqueous solution or suspension containing soluble nitrogen. In
some examples, the soluble nitrogen in a liquid fertilizer includes an organic source of nitrogen
such as urea, or urea derived from anhydrous ammonia (such as a solution of urea and
ammonium nitrate (UAN)). Aqua ammonia (20-32% anhydrous ammonia) can also be used. In
other examples, the soluble nitrogen in a liquid fertilizer includes nitrogen-containing inorganic
salts such as ammonium hydroxide, ammonium nitrate, ammonium sulfate, ammonium
pyrophosphate, ammonium thiosulfate or combinations of two or more thereof. In some
embodiments the liquid fertilizer includes a non-naturally occurring nitrogen source (such as
ammonium pyrophosphate or ammonium thiosulfate) and/or other non-naturally occurring
components.
Common liquid non-natural fertilizer blends are specified by their content of nitrogenphosphate-
potassium (N-P-K percentages) and include addition of other components, such as
sulfur or zinc. Examples of human-made blends include 10-34-0, 10-30-0 with 2% sulfur and
0.25% zinc (chelated), 11-37-0, 12-30-0 with 3% sulfur, 2-4-12, 2-6-12, 4-10-10, 3-18-6, 7-22-
5, 8-25-3, 15-15-3, 17-17-0 with 2% sulfur, 18-18-0, 18-18-0 with 2% sulfur, 28-0-0 UAN, 9-
27-0 with 2% sulfur and potassium thio-sulfate.
Microbe: A microorganism, including but not limited to bacteria, archaebacteria, fungi,
and algae (such as microalgae). In some examples, microbes are single-cellular organisms (for
example, bacteria, cyanobacteria, some fungi, or some algae). In other examples, the term
microbes includes multi-cellular organisms, such as certain fungi or algae (for example,
multicellular filamentous fungi or multicellular algae).
Microbial composition: A composition (which can be solid, liquid, or at least partially
both) that includes at least one microbe (or a population of at least one microbe). In some
examples, a microbial composition is one or more microbes (or one or more populations of
microbes) in a liquid medium (such as a storage, culture, or fermentation medium), for example,
as a suspension in the liquid medium. In other examples, a microbial composition is one or
more microbes (or one or more populations of microbes) on the surface of or embedded in a
solid or gelatinous medium (including but not limited to a culture plate), or a slurry or paste.
Microbial consortium: A mixture, association, or assemblage of two or more microbial
species, which in some instances are in physical contact with one another. The microbes in a
consortium may affect one another by direct physical contact or through biochemical
interactions, or both. For example, microbes in a consortium may exchange nutrients,
metabolites, or gases with one another. Thus, in some examples, at least some of the microbes
in a consortium may be metabolically interdependent. Such interdependent interactions may
change in character and extent through time and with changing culture conditions.
P. Microbial Consortia and Compositions
Disclosed herein are several microbial consortia. An exemplary microbial consortium of
the present disclosure was deposited with the American Type Culture Collection (ATCC,
Manassas, VA) on November 25, 2014, and assigned deposit number PTA-121755, referred to
herein as A1006. The A1006 consortium includes at least Bacillus spp., Pseudomonas spp.,
Lactobacillus spp., Desulfococcus spp., Desulfotomaculum spp., Marinobacter spp.,
Nitrosopumilus spp., Ruminococcus spp., Leptospirillum spp., Halorhabdus spp., Clostridium
spp., Xenococcus spp., Cytophaga spp., Candidatus spp., Microbacterium spp., Sporosarcina
spp., Lysinibacillus spp., Nesterenkonia spp., Agrococcus spp., Sphingomonas spp.,
Micrococcus spp., Paenibacillus spp., Acremonium spp., Leucobacter spp., Brevundimonas spp.,
Rhizobium spp., Chitinophaga spp., Brevibacillus spp., Virgibacillus spp., Rummeliibacillus
spp., Staphylococcus spp., and Oceanobacillus spp. detected in A1006 by microarray analysis
and/or 16 rDNA sequencing. Also disclosed herein are consortia or microbial compositions
including two or more (such as 2 or more, 5 or more, 10 or more, 20 or more, or 50 or more) or
all of the microbes in A1006. In some embodiments, a microbial composition disclosed herein
is a defined composition, for example a composition including specified microbial species and
optionally, additional non-microbial components (including but not limited to, salts, trace
elements, chitin, chitosan, glucosamine, and/or amino acids).
As discussed below, the identity of at least some microbes present in A1006 was
determined using microarray analysis (Example 3) and/or 16S rDNA sequencing (Example 5).
Additional techniques for identifying microbes present in a microbial mixture or consortium are
known to one of ordinary skill in the art, including sequencing or PCR analysis of nucleic acids,
such as 16S rDNA, from individual microbial colonies grown from within the consortium or
mixture. Additional techniques for identifying microbes present in a microbial mixture or
consortium also include 1) nucleic acid-based methods which are based on the analysis and
differentiation of microbial DNA (such as DNA microarray analysis of nucleic acids,
metagenomics or in situ hybridization coupled with fluorescent-activated cell sorting (FACS)),
2) biochemical methods which rely on separation and identification of a range biomolecules
including fatty acid methyl esters analysis (FAME), Matrix-assisted laser desorption ionizationtime
of flight (MALDI-TOF) mass spectrometry analysis, or cellular mycolic acid analysis by
High Performance Liquid Chromatography (MYCO-LCS) analysis, and 3) microbiological
methods which rely on traditional tools (such as selective growth and microscopic examination)
to provide more general characteristics of the community as a whole, and/or narrow down and
identify only a small subset of the members of that community.
In some examples, microbes in a mixture or consortium are separated (for example using
physical size and/or cell sorting techniques) followed by deep DNA or full genome sequencing
of the resulting microbes (or subgroups or subpopulations of microbes). Use of a different
microarray or use of other identification techniques may identify presence of different microbes
(more, fewer, or different microbial taxa or species) than the microarray analysis performed on
A1006 described herein, due to differences in sensitivity and specificity of the analysis
technique chosen. In addition, various techniques (including microarray analysis or PCR DNA
analysis) may not detect particular microbes (even if they are present in a sample), for example
if probes capable of detecting particular microbes are not included in the analysis. In addition,
one of ordinary skill in the art will recognize that microbial classification and naming may
change over time and result in reclassification and/or renaming of microbes.
In other embodiments the disclosed microbial consortia or compositions include, consist
essentially of, or consist of 2 or more (such as 5 or more, 10 or more, 15 or more, 20 or more, or
all) of the microbes listed in Table 1.
Table 1. Microbes

In some embodiments, the microbial composition includes an increased amount of
particular microbes compared to A1006. For example, culture of A1006 with liquid fertilizer
(for example, as described in Example 5) leads to an increase in the amount of one or more of
Bacillus spp. {e.g., one or more of Bacillus amyloliquefaciens, Bacillus pocheonensis, or
Bacillus clausii), Microbacterium spp. (e.g., Microbacterium testaceum), Lysinibacillus spp.
(e.g., Lysinibacillus sphaericus), Sporosarcina spp., Nesterenkonia spp., Agrococcus spp. (e.g.,
Agrococcus terreus), Acremonium spp. (e.g., Acremonium bacillisporum), Sphingomonas spp.,
Micrococcus spp., Paenibacillus spp., Leucobacter spp., Brevundimonas spp., Rhizobium spp.,
Chitinophaga spp., Brevibacillus spp., Virgibacillus spp., Rummeliibacillus spp.,
Staphylococcus spp., or Oceanobacillus spp. in the microbial composition. In some examples,
the microbial composition includes at least about 10% more of one or more of Bacillus spp.
(e.g., one or more of Bacillus amyloliquefaciens, Bacillus pocheonensis, or Bacillus clausii),
Microbacterium spp. (e.g., Microbacterium testaceum), Lysinibacillus spp. (e.g., Lysinibacillus
sphaericus), Sporosarcina spp., Nesterenkonia spp., Agrococcus spp. (e.g., Agrococcus terreus),
Acremonium spp., Sphingomonas spp., Micrococcus spp., Paenibacillus spp., Leucobacter spp.,
Brevundimonas spp., Rhizobium spp., Chitinophaga spp., Brevibacillus spp., Virgibacillus spp.,
Rummeliibacillus spp., Staphylococcus spp., or Oceanobacillus spp. compared to A1006.
The consortia or compositions can optionally include one or more additional microbes.
Additional microbes include, but are not limited to one or more of Deinococcus spp.,
Azospirillum spp., Aquabacterium spp., Acetobacter spp. (e.g., Acetobacter aceti), Acidisoma
spp., Azotobacter spp. (e.g., Azotobacter vinelandii), Treponema spp. (e.g., Treponema
primitia), Bradyrhizobium spp., Lactococcus spp., Leptolyngbya spp., Paenibacillus spp. (e.g.,
Paenibacillus amyloticus), Pediococcus (e.g., Pediococcus pentosceus), Proteus spp. (e.g.,
Proteus vulgaris), Rhizobium (e.g., Rhizobium japonicus), Rhodoferax spp., Streptomyces spp.,
Streptococcus spp., Trichoderma spp. (e.g., Trichoderma harzianum), Microcoleus spp.,
Micrococcus spp. (e.g., Micrococcus luteus), Nitrobacter spp., Nitrosomonas spp., Nitrospira
spp., Actinomyces spp., Devosia spp., Acetobacter spp., Brevibacterium spp., Methanosaeta
spp., Saccharomyces spp. (e.g., Saccharomyces cerevisiae), Penicillium spp. (e.g., Penicillium
roqueforti), Monascus (e.g., Monascus ruber), Aspergillus spp. (e.g., Aspergillus oryzae),
Arthrospira spp. (e.g., Arthrospira platensis), and Ascophyllum spp. (e.g., Ascophyllum
nodosum). Suitable additional microbes can be identified by one of skill in the art, for example,
based on characteristics desired to be included in the consortia or compositions.
The disclosed microbial consortia or compositions may include one or more further
components in addition to the microbes, including by not limited to salts, metal ions, and/or
buffers (for example, one or more of KH2PO4, K2HPO4, CaCb, MgS0 4 > FeCb, NaMo0 4, and/or
Na2Mo04), trace elements (such as sulfur, sulfate, sulfite, copper, or selenium), vitamins (such
as B vitamins or vitamin K), sugars (such as sucrose, glucose, or fructose), chitin, chitosan,
glucosamine, protein, and/or amino acids. Additional components that may also be included in
the compositions include HYTb, HYTc, and/or HYTd, one or more fertilizers (e.g., liquid
fertilizer), one or more pesticides, one or more fungicides, one or more herbicides, one or more
insecticides, one or more plant hormones, one or more plant elicitors, or combinations of two or
more of these components.
In some embodiments, the microbial consortia, or a composition including five or more
microbial species in the microbial consortia described herein are in a liquid medium (such as a
culture or fermentation medium) or inoculum. In other embodiments, the microbial consortia or
composition including five or more microbial species listed in Table 1 are present on a solid or
gelatinous medium (such as a culture plate) containing or supporting the microbes.
In yet other embodiments, the microbial consortia or composition including five or more
microbial species are present in a dry formulations, such as a dry powder, pellet, or granule. Dry
formulations can be prepared by adding an osmoprotectant (such as a sugar, for example,
trehalose and/or maltodextrin) to a microbial composition in solution at a desired ratio. This
solution is combined with dry carrier or absorptive agent, such as wood flour or clay, at the
desired concentration of microbial composition (such as 2-30%, for example, 2.5-10%, 5-15%,
7.5-20%, or 15-30%). Granules can be created by incorporating clay or polymer binders that
serve to hold the granules together or offer specific physical or degradation properties. Granules
can be formed using rotary granulation, mixer granulation, or extrusion, as a few possible
methods. Additional methods for preparing dry formulations including one or more microbial
species are known to one of ordinary skill in the art, for example as described in Formulation of
Microbial Biopesticides: Beneficial Microorganisms, Nematodes and Seed Treatments, Burges,
ed., Springer Science, 1998; Bashan, Biotechnol. Adv. 16:729-770, 1998; Ratul et al, Int. Res. J.
Pharm. 4:90-95, 2013.
In some examples, compositions including the microbes or microbial consortia may be
maintained at a temperature supporting growth of the microbe(s), for example at about 25-45°C
(such as about 30-35°C, about 30-40°C, or about 35-40°C). In other examples, the compositions
are stored at temperatures at which the microbe(s) are not growing or are inactive, such as less
than 25°C (for example, 4°C, -20°C, -40°C, -70°C, or below). One of skill in the art can
formulate the compositions for cold storage, for example by including stabilizers (such as
glycerol). In still further examples, the compositions are stored at ambient temperatures, such as
about 0-35°C (for examples, about 10-30°C or about 15-25°C).
PI. Biodegradation Processes
The disclosed microbial consortia or compositions can be used to degrade biological
materials, such as chitin-rich materials, for example, aquatic animals or aquatic animal byproducts,
insects, or fungi. Thus, in some embodiments, disclosed herein are methods including
mixing one or more of the disclosed microbial consortia or compositions with a chitincontaining
biological material to form a mixture, and fermenting the mixture. In some
embodiments, the methods also include separating the mixture into solid, aqueous, and
optionally, lipid fractions (FIG. 2).
In some embodiments, a biodegradation process disclosed herein includes mixing a
microbial consortium (such as A1006), a composition including some or all of the microbes in
A1006, or a composition including five or more of the microbial species in Table 1) with one or
more chitin-containing biological materials. Chitin-containing biological materials include, but
are not limited to, aquatic animals or aquatic animal by-products, insects, or fungi. In some
examples, the chitin-containing biological material is an aquatic animal, such as an aquatic
arthropod (for example, a member of Class Malacostraca). Aquatic arthropods for use in the
disclosed methods include shrimp, crab, lobster, crayfish, or krill. In some examples, the entire
aquatic animal (such as an aquatic arthropod) or aquatic animal by-products are used in the
biodegradation methods disclosed herein. Aquatic animal by-products include any part of an
aquatic animal, such as any part produced by processing of the aquatic animal. In some
examples, an aquatic animal by-product is all or a portion of an aquatic animal exoskeleton, such
as shrimp, crab, crayfish, or lobster shell. In other examples, an aquatic animal by-product is a
part of an aquatic animal, for example, shrimp cephalothoraxes.
In other examples, the chitin-containing biological material includes fungi, such as fungi
from Phylum Zygomycota, Basidiomycota, Ascomycota, or Deuteromycota. Particular
exemplary fungi include Aspergillus spp., Penicillium spp., Trichoderma spp., Saccharomyces
spp., and Schizosaccharomyces spp. Thus, baker, brewer, and distiller waste streams can
provide sources for chitin-containing biological material. In still further examples, the chitincontaining
biological material includes insects that contain chitin in their exoskeletons, such as
grasshoppers, crickets, beetles, and other insects. Byproducts of the processing of such insects
are also contemplated to be sources of chitin.
The chitin-containing biological material is mixed with a composition including the
microbes described in Section II above (such as the microbial consortium A1006 or other
consortium or composition described in Section II) to form a substantially homogeneous
mixture. In some examples, the chitin-containing biological material is ground, crushed,
minced, milled, or otherwise dispersed prior to mixing with the microbes or microbial consortia
described herein. In particular examples, the mixture contains about 10-50% (such as about 10-
20%, about 20-30%, about 30-40%, about 25-40%, for example about 25%, about 30%, about
35%, about 40%, about 45%, or about 50%) chitin-containing material (such as shrimp heads)
(w/v) in inoculum containing about 0.1-5% (such as about 0.1-1%, about 0.5-2%, about 1-2%,
about 2-3%, about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.8%, about 1%, about
1.25%, about 1.5%, about 1.75%, about 2%, about 2.5%, about 3%, about 4%, or about 5%)
microbes (v/v).
In some examples, the inoculum, chitin-containing biological material, and a sugar (or
other carbon source) are mixed together, for example by stirring or agitation. In other examples,
one or more of the microbes in the microbial composition or consortium is optionally activated
prior to mixing with the chitin-containing biological material and fermentation. Activation is
not required for the methods disclosed herein. Adjustments to the time and/or temperature of
the fermentation can be made by one of skill in the art, depending on whether the microbes are
activated prior to fermentation. Activation of the microbial composition can be by incubating an
inoculum of the microbes with a carbon source (such as a sugar, for example, glucose, sucrose,
fructose, or other sugar) at a temperature and for a sufficient period of time for the microbes to
grow. In some examples, an inoculum of the microbes (such as a microbial consortium or
composition described herein) has a concentration of about 0.05-5% v/v (for example, about 0.5-
5%, about 0.5-2%, about 1-2%, or about 2-3%) in a liquid medium. The inoculum is diluted in a
solution containing about 0.1-1% sugar (for example, about 0.1-0.5%, about 0.1-0.3%, about
0.2-0.6%, or about 0.5-1%, such as about 0.1%, about 0.2%, about 0.3%, about 0.4%, about
0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1%) and incubated at ambient
temperatures, for example about 20-40°C (such as about 20°C, about 25°C, about 30°C, about
35°C, or about 40°C) for about 1-5 days (such as about 24 hours, about 48 hours, about 72
hours, about 96 hours, or about 120 hours). In other examples, activation of the microbial
composition can be activated by incubating an inoculum of the microbes at a temperature and
for a sufficient period of time for the microbes to grow, for example, incubation at about 20-
40°C (such as about 25-35°C) for 12 hours to 5 days (such as 1-4 days or 2-3 days). In some
non-limiting examples, the microbes are considered to be activated when the culture reaches an
optical density of >0.005 at 600 nm.
After mixing of the chitin-containing biological material and the microbes or microbial
consortium (which are optionally activated), the mixture is fermented. In some examples, the
pH of the mixture is measured prior to fermentation. The pH is adjusted to a selected range
(e.g, pH about 3 to about 4 or about 3.5 to 4), if necessary, prior to fermentation. The mixture is
incubated at a temperature of about 20-40°C (for example, about 30°-36°C, such as about 30°C,
about 3 1°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about
38°C, about 39°C, or about 40°C) for about 1-30 days (such as about 3-28 days, about 7-21
days, about 3, 5, 7, 10, 14, 16, 20, 24, 28, or 30 days). The mixture is agitated periodically (for
example, non-continuous agitation). In some examples, the mixture is agitated for a period of
time every 1-7 days, for example every 1, 2, 3, 4, 5, 6, or 7 days. In some non-limiting
examples, the fermentation proceeds until the titratable acidity (TTA) is about 3-5% and the pH
is about 4-5.
Following the fermentation, the resulting fermented mixture is separated into at least
solid and liquid fractions. In some examples, the fermentation is passed from the tank to settling
equipment. The liquid is subsequently decanted and centrifuged. In one non-limiting example,
the fermented mixture is centrifuged at 1250 rpm (930xg) for 15 minutes at about 5°C to obtain
liquid and lipid (e.g., pigment) fractions. The liquid (or aqueous) fraction obtained from the
biodegradation process can be stored at ambient temperature. In some non-limiting examples, a
sugar is added to the liquid fraction, for example at 1-10% v/v.
The liquid fraction may include components such as protein, amino acids, glucosamine,
trace elements (such as calcium, magnesium, zinc, copper, iron, and/or manganese), and/or
enzymes (such as lactic enzymes, proteases, lipases, and/or chitinases). In some non-limiting
examples, the liquid fraction contains (w/v) about 1-5% total amino acids, about 3-7% protein,
about 0.1-2% nitrogen, less than about 0.2% phosphorus, about 0.5-1% potassium, about 4-8%
carbon, about 0.2-1% calcium, less than about 0.2% magnesium, less than about 0.2% sodium,
and/or about 0.1-0.4% sulfur. In additional non-limiting examples, the liquid fraction includes
about 0.01-0.2% glucosamine (for example, about 0.1% or less). The liquid fraction also may
contain one or more microbes {e.g., from the inoculum used to start the fermentation process)
and/or trace amounts of chitosan or chitin. The liquid fraction is in some examples referred to
herein as "HYTb."
The solid fraction obtained from the biodegradation process contains chitin (for example,
about 50-70% or about 50-60% chitin). The solid fraction may also contain one or more of trace
elements (such as calcium, magnesium, zinc, copper, iron, and/or manganese), protein or amino
acids, and/or one or more microbes from the inoculum used to start the fermentation process.
The solid fraction is in some examples referred to herein as "HYTc." HYTc is optionally
micronized to form micronized chitin and residual chitin. In some non-limiting examples, the
solid fraction contains (w/v) about 9-35% total amino acids, about 30-50% crude protein, about
5-10% nitrogen, about 0.3-1% phosphorus, less than about 0.3% potassium, about 35-55%
carbon, about 0.5-2% calcium, less than about 0.1% magnesium, about 0.1-0.4% sodium, and/or
about 0.2-0.5% sulfur.
In some examples, a lipid fraction is also separated from the solid and liquid fractions.
The lipid fraction is the upper phase of the liquid fraction. The lipid fraction contains
compounds such as sterols, vitamin A and/or vitamin E, fatty acids (such as DHA and/or EHA),
and in some examples, carotenoid pigments (for example, astaxanthin). The lipid fraction may
be used for a variety of purposes, including but not limited to production of cosmetics or
nutritional products.
In additional embodiments, chitin is fermented with a microbial consortium (such as
A1006 or some or all of the microbes in A1006) or a composition containing five or more of the
microbial species in Table 1. In some examples chitin (such as HYTc, or micronized and/or
residual chitin produced as described above) is mixed with a microbial consortium or
composition containing microbes described herein and protein hydrolyzate (e.g., HYTb), and
fermented to form a fermented mixture. At least a portion of the chitin in the starting mixture is
digested as a result of the fermentation. In some examples, the mixture is incubated at a
temperature of about 20-40°C (for example, about 30°-35°C, such as about 30°C, about 31°C,
about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about
39°C, or about 40°C) for about 1 day to 30 days (such as about 2-28 days, about 4-24 days,
about 16-30 days, about 10-20 days, or about 12-24 days). In some examples, the mixture is
agitated periodically (for example, non-continuous agitation). In other examples, the mixture is
continuously agitated. In one non-limiting example, the mixture is agitated for about 1-12 hours
daily (such as about 2-8 hours or about 4-10 hours). The pH of the fermentation mixture may be
monitored periodically. In some examples, the pH is optionally maintained at about 4-5. In
some examples, the fermentation proceeds until Total Titratable Acidity (TTA) is at least about
1-10% (such as about 2-8%, about 4-8%, or about 5-10%).
Following the fermentation, the resulting fermented mixture is separated into at least
solid and liquid fractions, for example by decanting, filtration, and/or centrifugation. The liquid
fraction resulting from fermentation of HYTb and chitin with the microbial composition is in
some examples referred to herein as "HYTd." In some non-limiting examples, the liquid
fraction contains (w/v) about 0.5-2% total amino acids, about 3-7% protein, about 0.5-1%
nitrogen, less than about 0.1% phosphorus, about 0.4-1% potassium, about 3-7% carbon, less
than about 0.5% calcium, less than about 0.1% magnesium, less than about 0.3% sodium, and/or
about less than about 0.3% sulfur. In addition, HYTd contains less than about 50% chitin (such
as less than about 45%, less than about 40%, less than about 35%, or less than about 30% chitin)
and less than 2% glucosamine (such as less than about 1.5% or less than about 1%
glucosamine). In other examples, HYTd contains about 25-50% chitin and about 0.5-2%
glucosamine.
IV. Processes for Treating Soil, Plants, and/or Seeds
The disclosed microbial consortia, compositions containing microbes, and/or products
disclosed herein (such as HYTb, HYTc, and/or HYTd) can be used to treat soil, plants, or plant
parts (such as roots, stems, foliage, seeds, or seedlings). In some examples, treatment with the
microbial consortia, compositions containing microbes, and/or products improve plant growth,
improve stress tolerance and/or increase crop yield.
In some embodiments the methods include contacting soil, plants (such as plant foliage,
stems, roots, seedlings, or other plant parts), or seeds with a consortium (such as A1006) or a
composition including the microbes present in one or more of the disclosed microbial consortia
or compositions. The methods may also include growing the treated plants, plant parts, or seeds
and/or cultivating plants, plant parts, or seeds in the treated soil.
The microbes are optionally activated before application. In some examples, activation
of the microbes is as described in Section III, above. In other examples, the microbes are
activated by mixing 100 parts water and 1 part microbial consortium or composition and
incubating at about 15-40°C (such as about 20-40°C, about 15-30°C, or about 25-35°C) for
about 12 hours-14 days (such as about 1-14 days, 3-10 days, 3-5 days, or 5-7 days). The
activation mixture optionally can also include 1part HYTb, if the microbial consortium or
composition is to be applied in combination with HYTb.
In other embodiments, the methods include contacting soil, plants (or plant parts), or
seeds with a product of the disclosed microbial consortia or compositions, such as HYTb,
HYTc, HYTd, or combinations thereof. In still further embodiments, the methods include
contacting soil, plants, or seeds with a disclosed microbial consortium or composition including
the disclosed microbes and one or more of HYTb, HYTc, and HYTd (such as one, two, or all of
HYTb, HYTc, and HYTd). HYTb, HYTc, and/or HYTd may be separately applied to the soil,
plants (or plant parts), and/or seeds, for example sequentially, simultaneously, or substantially
simultaneously with the disclosed microbial consortia or compositions containing microbes.
In some examples, the methods further include contacting the soil, plants (or plant part),
or seeds with one or more additional components including but not limited to chitin, chitosan,
glucosamine, protein, amino acids, liquid fertilizer, one or more pesticides, one or more
fungicides, one or more herbicides, one or more insecticides, one or more plant hormones, one
or more plant elicitors, or combinations of two or more thereof. The additional components may
be included in the composition including the microbes or in the microbial consortia disclosed
herein, or may be separately applied to the soil, plants (or plant parts), and/or seeds, for example
sequentially, simultaneously, or substantially simultaneously with the disclosed microbial
consortia or compositions containing microbes.
In particular embodiments, a microbial consortium or composition is combined with a
liquid fertilizer (for example an aqueous solution or suspension containing soluble nitrogen). In
some examples, the liquid fertilizer includes an organic source of nitrogen such as urea, or a
nitrogen-containing inorganic salt such as ammonium hydroxide, ammonium nitrate, ammonium
sulfate, ammonium pyrophosphate, ammonium thiosulfate or combinations thereof. Aqua
ammonia (20-24.6% anhydrous ammonia) can also be used as the soluble nitrogen. In some
examples, the microbial consortium or composition is combined with the liquid fertilizer (for
example, mixed with the liquid fertilizer) immediately before use or a short time before use
(such as within 10 minutes to 24 hours before use, for example, about 30 minutes, 1 hour, 2
hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, or 24 hours before use).
In other examples, the microbial consortium or composition is combined with the liquid
fertilizer (for example mixed with the liquid fertilizer) at least 24 hours before use (such as 24
hours to 6 months, for example, at least 36 hours, at least 48 hours, at least 72 hours, at least 96
hours, at least one week, at least two weeks, at least four weeks, at least eight weeks, or at least
12 weeks before use).
In some examples, the amount of the composition(s) to be applied (for example, per acre
or hectare) is calculated and the composition is diluted in water (or in some examples, liquid
fertilizer) to an amount sufficient to spray or irrigate the area to be treated (if the composition is
a liquid, such as microbial consortia or compositions, HYTb, or HYTd). In other examples, the
composition can be mixed with diluted herbicides, insecticides, pesticides, or plant growth
regulating chemicals. If the composition to be applied is a solid (such as a dry formulation of
microbes, HYTc, chitin, glucosamine, chitosan, or amino acids), the solid can be applied directly
to the soil, plants, or plant parts or can be suspended or dissolved in water (or other liquid) prior
to use. In some examples, HYTc is dried and micronized prior to use.
The disclosed microbial compositions (alone or in combination with other components
disclosed herein, such as HYTb, HYTc, and/or HYTd) can be delivered in a variety of ways at
different developmental stages of the plant, depending on the cropping situation and agricultural
practices. In some examples, a disclosed microbial composition and HYTb are mixed and
diluted with liquid fertilizer and applied at the time of seed planting at a rate of 0.5 to 1 to 2
liters each per acre, or alternatively are applied individually. In other examples, a disclosed
microbial composition and HYTb are mixed and diluted and applied at seed planting, and also
applied to the soil near the roots at multiple times during the plant growth, at a rate of 0.5 to 1 to
2 liters each per acre, or alternatively are applied individually. In still further examples, a
disclosed microbial composition and HYTb are diluted and delivered together through drip
irrigation at low concentration as seedlings or transplants are being established, delivered in
flood irrigation, or dispensed as a diluted mixture with nutrients in overhead or drip irrigation in
greenhouses to seedlings or established plants, or alternatively are applied individually. In
additional examples, a disclosed microbial composition is added to other soil treatments in the
field, such as addition to insecticide treatments, to enable ease-of-use. In other examples, such
as greenhouses, a disclosed microbial composition and HYTb are used individually or together,
combined with liquid fertilizer (such as fish fertilizer) and other nutrients and injected into
overhead water spray irrigation systems or drip irrigation lines over the course of the plant's
growth. In one greenhouse example, a disclosed microbial composition and HYTb are used
together, for example, diluted and applied during overhead irrigation or fertigation at a rate of
0.25 to 1 liter at seedling germination, followed by 0.25 to 1 liter mid-growth cycle with
fertigation, and final 0.25 to 1 liter fertigation 5-10 days end of growth cycle.
In some embodiments, a disclosed microbial composition or consortium and HYTb are
applied together or individually (for example sequentially) to promote yield, vigor, typeness,
quality, root development, and stress tolerance in crops. In one specific example where the crop
is corn, 1 to 2 L/acre microbial composition is added in-furrow with liquid fertilizer at seed
planting, or applied as a side dress during fertilization after V3 stage, followed by 0.5 to 2 L/acre
of HYTb as a foliar spray after V5 stage, added and diluted with herbicides, foliar pesticides,
micronutrients, or fertilizers.
In another specific example where the crop is potato, 1 to 3 L/acre of microbial
composition is diluted and used either alone or with 1 to 3 L/acre of HYTb at tuber planting; this
can be followed by subsequent soil applications of the microbial composition and HYTb before
tuberization, either alone (e.g., sequentially) or together. After plant emergence, potato foliar
applications of HYTb at 1 to 2 L/acre can be applied, either diluted alone or mixed with
herbicide, foliar pesticide, micronutrient, or fertilizer treatments, and applied during the growing
season one time, two times, three times, four times, or more.
In yet another specific example where the crop is cotton, 1 to 2 L/acre of microbial
composition is applied m-furrow at planting, as a side dress, or 2x2 (2 inches to side and 2
inches below seed), with or without fertilizer. At first white cotton bloom, foliar treatments of
0.5 to 2 L/acre HYTb can be applied, diluted alone or combined with other nutrient, herbicide,
or pesticidal treatments.
In another particular example where the crop is wheat, the microbial composition ( 1 to 2
L/acre) is applied after winter dormancy (S4 stage) and HYTb applied foliarly (0.5 to 2 L/acre;
S4 to S10 stage).
In an example where the crop is sugarcane, one application method uses a disclosed
microbial composition and HYTb at 2 to 4 L/acre each, applied to the soil during cane planting
or as a side dress, with foliar HYTb applied at 1 to 2 L/acre, mixing with water or fertilizers or
micronutrients.
HYTb can be used alone as a foliar treatment in all crops to improve traits such as plant
stress tolerance, vegetative vigor, harvest quality and yield. In an example where the crop is
corn, HYTb can be applied at ½ to 1 L/acre, one or multiple times, mixing with water or
pesticides or herbicides. In another example, HYTb can be used to treat wheat as a foliar spray,
mixed with water or pesticides or herbicides, at a rate of ½ to 1 L/acre, applying one or multiple
times.
In all crops, HYTc may be added to the soil at a rate of about 0.5-2 kg/acre (such as
about 0.5 kg/acre, about 1 kg/acre, about 1.5 kg/acre, or about 2 kg/acre) at the time of crop
establishment or planting. In other examples, HYTc is added to a drip irrigation solution of a
disclosed microbial composition and HYTb or is added to fertilization applications containing a
disclosed microbial composition and HYTb in greenhouses, such as the examples above.
In additional embodiments, HYTd (alone or in combination with the microbes or other
components disclosed herein) is used at about 1-20 L/hectare (such as about 1-15 L/hectare,
about 3-10 L/hectare, or about 3-5 L/hectare). In other examples, HYTd (alone or in
combination with the microbes or other components disclosed herein) is used as a seed treatment
to enhance crop yield and performance (for example, about 1-10 L/kg seed, such as about 1-3
L/kg, about 3-5 L/kg, or about 5-10 L/kg). Alternatively, HYTd can be used in the soil (alone or
in combination with the microbes or other components disclosed herein) at about 1-3 L/hectare
to increase plant growth, for example to help plants remain productive under conditions of
stress.
In some examples, treatment of soil, seeds, plants, or plant parts with a composition
comprising the microbes in a disclosed microbial consortium increases plant growth (such as
overall plant size, amount of foliage, root number, root diameter, root length, production of
tillers, fruit production, pollen production, or seed production) by at least about 5% (for
example, at least about 10%, at least about 30%, at least about 50%, at least about 75%, at least
about 100%, at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-
fold, or more). In other examples, the disclosed methods result in increased crop production of
about 10-75% (such as about 20-60% or about 30-50%) compared to untreated crops. Other
measures of crop performance include quality of fruit, yield, starch or solids content, sugar
content or brix, shelf-life of fruit or harvestable product, production of marketable yield or target
size, quality of fruit or product, grass tillering and resistance to foot traffic in turf, pollination
and fruit set, bloom, flower number, flower lifespan, bloom quality, rooting and root mass, crop
resistance to lodging, abiotic stress tolerance to heat, drought, cold and recovery after stress,
adaptability to poor soils, level of photosynthesis and greening, and plant health. To determine
efficacy of products, controls include the same agronomic practices without addition of
microbes, performed in parallel.
The disclosed methods can be used in connection with any crop (for example, for direct
crop treatment or for soil treatment prior to or after planting). Exemplary crops include, but are
not limited to alfalfa, almond, banana, barley, broccoli, canola, carrots, citrus and orchard tree
crops, corn, cotton, cucumber, flowers and ornamentals, garlic, grapes, hops, horticultural
plants, leek, melon, oil palm, onion, peanuts and legumes, pineapple, poplar, pine and woodbearing
trees, potato, raspberry, rice, sesame, sorghum, soybean, squash, strawberry, sugarcane,
sunflower, tomato, turf and forage grasses, watermelon, wheat, and eucalyptus.
The following examples are provided to illustrate certain particular features and/or
embodiments. These examples should not be construed to limit the disclosure to the particular
features or embodiments described.
Example 1
Microbial Consortium A1006
This example describes production of microbial consortium A1006.
A1006 was produced from a seed batch of microbes that originally were derived from
fertile soils and additional microbes (such as Bacillus spp.) (see, e.g., U.S. Pat. No. 8,748,124,
incorporated herein by reference). The "seed" culture was mixed with a suspension containing
5.5% w/w whey protein and 1.2% w/w yogurt in water ("C vat") and a suspension containing
0.1% w/w spirulina and 0.1% w/w kelp extract in water ("A vat"). The A vat and C vat
suspensions were each individually prepared 3 days before mixing with the seed culture and
incubated at ambient temperature. The seed culture, C vat, and A vat were mixed at a
proportion of about 81:9:9. After mixing, a suspension of additional components containing
about 70% v/v molasses, 0.5% v/v HYTb, 0.003% w/v Arabic gum, and 0.02% w/v brewer's
yeast (S. cerevisiae) were mixed with the mixture of the seed culture, C vat, and A vat, and
additional water at a ratio of about 16:34:50. The mixture was fermented for about 7 days at
ambient temperature (about 19-35°C). After 7 days, the tanks were aerated for 30 minutes every
other day. Additional water was added (about 10% more v/v) and fermentation was continued
under the same conditions for about 10 more days. Additional water was added (about 4% more
v/v) and fermentation was continued for about 7 more days, at which time samples were
collected for analysis and deposit with the ATCC. Al 006 was subsequently stored in totes at
ambient temperature.
Example 2
Analysis of Microbes in A1006 by Plating
This example describes analysis of microbes present in A1006 by replicate plating under
aerobic and anaerobic conditions.
Samples (50 mL) were collected from an aerated tote of A1006 (stirred with a stainless
steel mixing paddle at 120 rpm for 8 minutes) using a sanitized handheld siphon drum pump.
On day 1, the sample was vortexed (e.g., 60 seconds at 2000 rpm) to ensure even distribution of
microbes. In a tube with 9.8 mL sterile water, 0.1 mL of A1006 sample and 0.1 mL of HYTb
were added (10 2 dilution). The tube was incubated at 35°C for 72 hours without shaking. After
72 hours (day 3), the tube was briefly vortexed and a series of 10-fold dilutions in sterile water
was prepared 10 3 to 10 9 dilutions).
Each dilution was plated (100 pL) on a Nutrient Agar plate (for aerobic microorganism
culture) and a Standard Methods Agar plate (for anaerobic microorganism culture), with 3
replicates for each. Nutrient Agar plates were cultured at 27°C for 48 hours. Standard Methods
Agar plates were incubated at 35°C for 72 hours in an anaerobic chamber. After the incubation,
for each culture, a dilution that yielded less than 100 colonies was selected. For the selected
dilution all of the colonies on each of the replicate plates were counted and colony forming units
(CFU)/mL was calculated. A1006 plating showed 9.0 x 107 CFU/mL under aerobic conditions
and 1.4 x 107 CFU/mL under anaerobic conditions.
Example 3
Analysis of Microbes in A1006 by Microarray
This example describes microarray analysis of microbes present in A1006.
A sample of A1006 was analyzed by Second Genome (South San Francisco, CA) using
the G3 PhyloChip™ Assay. DNA was isolated from the sample using PowerSoil® DNA
isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA) according to the manufacturer's
instructions. 16S rRNA was amplified (35 PCR cycles) using Genes were amplified using the
degenerate forward primer 27F.1 (AGRGTTTGATCMTGGCTCAG; SEQ ID NO: 1) and the
non-degenerate reverse primer 1492R (GGTTACCTTGTTACGACTT; SEQ ID NO: 2). The
amplification products were concentrated using a solid-phase reversible immobilization method
and quantified by electrophoresis using an Agilent 2100 Bioanalyzer®. PhyloChip Control
Mix™ was added to each amplified product. The amplicons were fragmented, biotin labeled,
and hybridized to the PhyloChip™ G3 array, which includes > 1.1 million probes targeting about
55,000 individual microbial taxa, with multiple proves per operational taxonomic unit (OTU).
The arrays were washed, stained, and scanned using a GeneArray® scanner (GeneChip®
Microarray Analysis Suite, Affymetrix).
Approximately 330 billion molecules were assayed and analyzed using Second
Genome's PhyloChip processing software. A series of perfect match (PM) and mis-match
(MM) probes sets gave off a florescence intensity (FI) which were captured as pixels in an
image and collected as an integer value. The software then made adjustments for background
florescence and noise estimation and rank-normalized the results. The results were then used as
input to empirical probe-set discovery. The empirical OUT tracked by a probe set was then
taxonomically annotated against the May 201 3 release of Greengenes 16S rRNA gene database
(greengenes.lbl.gov) from the combination of 8-mers contained in all probes of the set. The taxa
were then identified by the standard taxonomic name or with a hierarchical taxon identifier.
After the taxa were identified for inclusion in analysis, the values used for each taxasample
were populated in two distinct ways. In the first case, a relative abundance metric was
used to rank the abundance of each taxa relative to the others. The second case used a binary
metric or presence/absence score to determine whether each taxon was actually in the sample.
The data from the microarray analysis were also used to select microbes for inclusion in
the compositions described herein (such as the microbes listed in Table 1 and elsewhere herein.).
The microbes (taxa, genus, or species) were ranked in order of relative abundance and microbes
were selected based on desired characteristics.
Example 4
Analysis of Microbes in A1006 by Sequencing
This example describes exemplary methods for analysis of microbes in A1006 by
sequencing 16S rDNA. One skilled in the art will appreciate that methods that deviate from
these specific methods can also be used for successful sequencing and analysis of microbes in
A1006.
Genomic DNA is extracted from a sample of A1006. 16S rDNA is amplified by PCR
and sequenced, for example using MICROSEQ ID microbial identification system (Applied
Biosystems/Life Technologies, Grand Island, NY). Sequencing data is analyzed, for example
using SHERLOCK DNA software (MIDI Labs, Newark, DE).
Example 5
Growth of Microbes in Nitrogen Fertilizer
This example describes selecting subpopulations of the microbial consortium using
different growth conditions, such as exposure to liquid fertilizers. This example also
demonstrates the tolerance of the microbes to high concentrations of nitrogen fertilizers and the
utility of combining the microbe consortium with fertilizers used in agriculture.
The microbial consortium was combined with either liquid urea-ammonia-nitrogen
fertilizer (UAN 32) or with ammonium polyphosphate (10-34-0; AP) fertilizer in a ratio of 90:1
(fertilizer:microbes) in 250 mL lidded growth containers and grown either at 4°C or 23°C.
Controls consisted of watenmicrobe dilutions (90:1) grown in parallel. Samples were cultured
without agitation for 28 days. At 7 day intervals, the sample was mixed to uniformity, a 1mL
aliquot recovered, serially diluted, plated onto standard growth media (Nutrient Agar/NA for
aerobic, Standard Methods Agar/SMA for anaerobic growth assessment), and grown under
either aerobic (27°C) or anaerobic (35°C) conditions for 48 or 72 hours, respectively. Growth
curves were performed in triplicate.
The growth rates for UAN 32 exposed microbes (UAN32) versus Control (CON) grown
at 4°C is illustrated in Figure 4A or UAN 32 exposed microbes (UAN) versus Control (CON)
grown at 23°C (Figure 4B). Marked differences in growth profile over the 28 day incubation
period were apparent between the two temperatures and in the recovery of aerobic or anaerobic
populations. At 4°C (FIG. 4A), aerobic populations in both UAN-exposed and Control samples
peaked at day 14 and slowly declined. The anaerobic growth patterns were different, with
Control growth peaking at day 21, whereas the UAN-exposed microbial growth declined
between day 7 and 14 and later recovered by day 28.
Growth at 23°C (FIG. 4B) showed strong growth of Control cultures over the 28 day
time course for aerobic populations; anaerobic growth flattened out by day 14. In contrast, both
the aerobic and anaerobic growth patterns for UAN-exposed cultures were similar and stayed
relatively fiat. Comparing the UAN-exposed populations with AP-exposed populations showed
a similar relatively flat growth profile for both aerobic and anaerobic bacteria (FIG. 4C).
Cleanly separated colonies were sent to MIDI Labs, Inc. (Newark, DE) for sequencing of
the 16S variable region ribosomal DNA for species identification (as described in Example 4).
Purified isolates were identified and are listed in Table 2. A species level match was assigned if
the %GD (generic difference) between the unknown and the closest match was less than the
approximate average %GD between species within that particular genetic family, which is
usually 1%. A genus level match was assigned when the sequence did not meet the
requirements for a species level match, but still clustered within the branching of a well-defined
genus. (1%< %GD <3%).
Table 2. Microbes identified by sequencing of colonies from UAN or AP exposed A1006.
Example 6
Biodegradation of Chitin-Containing Materials
This example describes exemplary methods for biodegradation of chitin-containing
biological materials using the microbial consortium Al 006. However, one skilled in the art will
appreciate that methods that deviate from these specific methods can also be used for successful
biodegradation of chitin-containing biological materials.
Shrimp by-products are obtained from shrimp processing plants and transported in
closed, chilled containers. After inspection of the raw material quality, the shrimp by-products
are homogenized to reduce particle size to about 3-5 mm. Pre-activated A1006 microbial
cultures (about 0.2-100 mL/L) and sucrose (about 5 g/L) are mixed with the homogenized
shrimp by-product (about 50 g/L) and agitated until the mixture is homogeneous. With
continuous agitation, the temperature is maintained at ambient temperature (about 19-35°C) and
the pH is adjusted to 3.5-4.0 with citric acid. The mixed ingredients are transferred into a
sanitized fermentation tank (25,000 L) and fermented at 30-36°C for 120 hrs. Agitation is
applied for 30 minutes at least two times a day. During the fermentation process, the pH is
monitored, and the total titratable acidity (TTA, %) is determined by titration with 0.1 N NaOH.
The fermentation is stopped when the TTA is about 3.5% and/or the pH is about 4-5.
The fermented cultures are fed to a continuous decanter. The separated solid layer from
the decanting step is subject to centrifugation to remove the lipid layer. The purified liquid
(HYTb) is mixed with sugar (such as molasses, 10% v/v), then stored in holding tanks or
dispensed to totes. The solid materials from the decanting step are dried with superheated air at
120°C until their moisture content is below 8%, then ground to 200 mesh. The dried product
(HYTc) is packaged in bags or sacks.
Example 7
Biodegradation of Chitin
This example describes exemplary methods for biodegradation of chitin using the
microbial consortium A1006. However, one skilled in the art will appreciate that methods that
deviate from these specific methods can also be used for successful biodegradation of chitin.
A1006 microbial culture is pre-activated with sugar (about 2.5 g/L) in a 10,000 L tank
for three days. The activated inoculum is mixed with protein hydrolysate such as HYTb (about
500 mL/L) and chitin (HYTc e.g., produced as described in Example 6). The mixture is gently
mixed for 1 hour to achieve complete homogenization. The mixture is fermented for 20 days at
ambient temperature (e.g., about 19-35°C) with agitation for about 8 hours daily and pH
monitoring (pH 4.0-5.0). Samples may be collected periodically, for instance every two days,
for quantification of glucosamine and optionally chitosan. After fermentation is complete, the
mixture is filtered through a filter that retains particles of 300 mesh, primarily the remaining
chitin. The filtrate is retained and bottled after product characterization.
Example 8
Treatment of Field Corn with Microbial Compositions
This example describes a representative method for obtaining increased corn crop yield,
using a microbial consortium. One skilled in the art will appreciate that methods that deviate
from these specific methods can also be used for increasing crop yield.
Treatment of field corn with a microbial composition similar to A1006, or with HYTb,
showed a strong increase in final harvestable yield. All agronomic practices of fertilization,
cultivation, weed control, and pest control, were identical and side-by-side for the microbial
composition- or HYTb-treated plots (Test) and control (Check) plots.
Trial 1 evaluated yield after the microbial composition was added to the typical nitrogen
side dress ( 1 L/acre microbial composition; 32 UAN liquid fertilizer; Test) compared to nontreated
control (Check), applied at V2 stage. In two large-scale, replicated strip trials ( 1 acre
total), yield in the Test strips were 8% to 10% higher than parallel control strips (Check) (FIG.
5A).
Trial 2 demonstrated that both in-furrow application and addition in the side dress were
equally effective for increasing corn yields. In a 1 acre strip trial, large plots were treated with
the microbial composition added in-furrow, during seed planting ( 1 L/acre) or at V2 stage as a
side dress (3 gal NPK liquid fertilizer, 1 liter micronutrient mix). Both application methods
showed Test strips had about a 5% increase in yield, about 10 Bu/acre compared to controls
(FIG. 5B). Adding a commercial blend of 10% humic acid/biostimulant to the Test (Actuate) infurrow
offered the same 5% yield as microbial composition addition alone compared to nontreated
control (FIG. 5B).
Trial 3 demonstrated that addition of nitrogen-stabilization products either unaffected or
slightly boosted the yield enhancing effect of the microbial composition in corn and further
validated the consistent boost in yield of the microbial composition delivered either in-furrow or
mixed in the side dress (FIG. 5C). In a 1 acre strip trial, both in-furrow and side dress
treatments offered a 3% yield boost (8 Bu/acre) over control (Check). Addition of Actuate
caused a slight yield increase (4% boost in yield, 9 Bu/acre higher than control). Addition of
nitrogen-stabilization products, Instinct or N-Kress, caused either no effect (modest 2.5% yield
boost for Instinct) or a slightly higher boost in yield (4.6% yield increase for N-Kress, 11
Bu/acre higher than control).
Trial 4 demonstrated that HYTb delivered in-furrow also boosted yield over control
plots. In a 20 acre trial, HYTb was added to the in-furrow fertilizer/nutrient mix ( 1 L/acre).
Compared to parallel control acreage (Check), HYTb-treated acres offered a 3.5% (7 Bu/acre)
yield increase (FIG. 5D).
Trial 5 demonstrated that, when evaluated in a replicated plot design trial, a single soil
inoculation of corn with the microbial composition at 1 L/acre in furrow at V6 stage, delivered
with 28% nitrogen fertilizer via drip irrigation, provided a 14% increase yield over the untreated
control across five replicated plots (FIG. 5E).
Trial 6 showed that HYTb, when used alone as a foliar treatment in corn, also provided a
9.5% yield increase when compared to the untreated control when tested in a randomized,
replicated plot design trial. HYTb was foliar sprayed over two applications of 1 L/acre each
application, at the V8 stage and VT stages (FIG. 5F).
Trial 7 was also a randomized and replicated plot design trial in corn, performed under
water stress conditions. In this study, the amount of irrigation was limited to 11 inches of water
versus the appropriately watered plots that received 17 inches of irrigation. A single 1 L/acre
treatment of microbial composition, delivered at stage V6 with 28% nitrogen fertilizer via drip
irrigation (Treated), produced a 38% yield increase over plots treated with fertilizer alone
(untreated Check). The harvest increase observed with microbial composition treatment
represents a potential of 31 Bu/acre higher yield (FIG. 5G).
Example 9
Treatment of Wheat with Microbial Compositions
This example describes a representative method for obtaining increased wheat crop
yield, using a microbial consortium. One skilled in the art will appreciate that methods that
deviate from these specific methods can also be used for increasing crop yield.
Treatment of wheat with a microbial composition prepared similarly to A1006, or with
HYTb, showed a strong increase in final harvestable yield. All agronomic practices of
fertilization, cultivation, weed control, and pest control, were identical and side-by-side for the
microbial composition- or HYTb-treated plots (Test) and control (Check) plots.
Trial 1 showed a strong increase in wheat yield promoted by soil application of the
microbial composition. In this 80 acre trial, the microbial composition was added at a rate of 1
L/acre to the top dress fertilizer mix at stage S4. Harvest yields demonstrated an 11% (10
Bu/acre) yield increase with use of the microbial composition (FIG. 6A).
Trial 2 compared three large trials in the same geographic area, totaling 271 acres of
microbial composition-treated (test) and 354 acres of parallel untreated wheat (control). All
trials were performed the same, with microbial composition ( 1 L/acre) added to the top dress
fertilizer mix and applied at wheat growth stage S4. Relative to parallel control acres on Hie
same farm, the treated wheat gave higher yields, ranging from an increase of 6% to 17% to 36%
higher yields, with a three farm average of about 16% increase in yield (FIG. 6B).
Trial 3 evaluated microbial composition and HYTb treatment of wheat in combination
and found that the combination enhanced yield. In a large pivot trial (129 acres), microbial
composition was applied pre-plant at a rate of 1 L/acre, incorporated with normal nutritional
program, and followed by pivot delivery of HYTb as a foliar spray (1 L/acre) plus herbicide at
wheat growth stage S6. Compared to untreated control (Check), the treated acreage gave a 10%
higher yield (14 Bu/acre) than control acreage (FIG. 6C). Further, typical wheat plants from the
treated plots had visibly more roots than untreated controls (FIG. 6D).
Example 10
Treatment of Tomato with A1006
This example describes a representative method for obtaining increased tomato crop
yield utilizing the A1006 microbial consortium. One skilled in the art will appreciate that
methods that deviate from these specific methods can also be used for increasing crop yield.
Treatment of tomato with A1006 showed a strong increase in final harvestable yield. All
agronomic practices of fertilization, cultivation, weed control, and pest control, were identical
and side-by-side for both the microbial composition-treated (Test) and control (Check) plots.
Trial 1 evaluated treatment of tomato applied at 1 L/acre with one application at
transplant (in transplant water) followed by application by drip irrigation every three weeks
(four times). In a 10 acre test plot compared to a 10 acre control plot, the treated acreage gave
about 8% higher yield than control (FIG. 7A).
Trial 2 evaluated treatment of tomato applied at 1 L/acre by drip irrigation every three
weeks (five times). In a 49.6 acre test plot compared to a 4.45 acre control plot, the treated
acreage gave about 9% higher yield than control (FIG. 7B).
Trial 3 evaluated treatment of tomato applied at 1 L/acre with one application at
transplant (in transplant water) followed by application by drip irrigation every three weeks
(three times). In a 15.6 acre test plot compared to a 73.2 acre control plot, the treated acreage
gave about 29% higher yield than control (FIG. 7C).
Trial 4 evaluated treatment of tomato applied at 1 L/acre with by drip irrigation every
three weeks (four times). In an 8.7 acre test plot compared to a 6.57 acre control plot, the treated
acreage gave decreased yield compared to control (FIG. 7D). However, the trial was affected by
severe disease pressure (Fusarium) which likely affected the outcome of the trial. In addition,
this trial was a relatively small plot size and also included different crop varieties in the
treatment.
Trial 5 evaluated treatment of tomato applied at 1 L/acre in combination with fertilizer
treatment. One application was at transplant with 8-7-7, followed by application by drip
irrigation every three weeks (three times) with UAN. In a 33.3 acre test plot compared to a
16.45 acre control plot, the treated acreage gave about 5% higher yield than control (FIG. 7E).
Example 11
Treatment of Sunflower with Microbial Compositions
This example describes a representative method for obtaining increased sunflower crop
yield, using a microbial consortium. One skilled in the art will appreciate that methods that
deviate from these specific methods can also be used for increasing crop yield.
Treatment of sunflower crop with a microbial composition prepared similarly to A1006
showed a strong increase in final harvestable yield. All agronomic practices of fertilization,
cultivation, weed control, and pest control, were identical and side-by-side for both the
microbial composition-treated (Test) and control (Check) plots.
This trial evaluated microbial composition treatment of sunflower applied at 1 L/acre by
drip irrigation 30 days and 60 days post-planting. In a 93.5 acre test plot compared to a 97. 13
acre control plot, the treated acreage gave about 50% higher yield than control (FIG. 8). In
addition, the treatment resulted in increased germination rates.
Example 12
Treatment of Rice with Microbial Compositions
This example describes a representative method for obtaining increased rice crop yield,
using a microbial consortium. One skilled in the art will appreciate that methods that deviate
from these specific methods can also be used for increasing crop yield.
Treatment of rice with a microbial composition prepared similarly to A1006 showed a
strong increase in final harvestable yield. All agronomic practices of fertilization, cultivation,
weed control, and pest control, were identical and side-by-side for both the microbial
composition-treated (Test) and control (Check) plots.
This trial evaluated microbial composition treatment of rice applied at 1 L/acre with aqua
ammonia. In a 61.8 acre test plot compared to a 100.7 acre control plot, the treated acreage gave
about 6% higher yield than control (FIG. 9).
Example 13
Treatment of Soybean with Microbial Compositions
This example describes a representative method for obtaining increased soybean crop
yield, using a microbial consortium. One skilled in the art will appreciate that methods that
deviate from these specific methods can also be used for increasing crop yield.
Treatment of soybean with a microbial composition prepared similarly to A1006, or with
HYTb, showed a strong increase in final harvestable yield. All agronomic practices of
fertilization, cultivation, weed control, and pest control, were identical and side-by-side for the
microbial composition- or HYTb-treated plots (Test) and control (Check) plots.
Trial 1 showed an increase in soybean yield promoted by application of HYTb at 1
L/acre, applied with fungicide. In two one acre tests, the treated acreage gave about 5%
increased yield compared to control (FIG. 10A).
Trial 2 evaluated microbial composition treatment or microbial composition plus HYTb
treatment of soybean applied at 1 L/acre by foliar and side dress application. The treated
acreage had reduced yield compared to control (FIG. 10B). However, the trial was affected by
small plot size combined with wildlife problems (deer nested and consumed the beans before
harvest).
Trial 3 showed an increase in soybean yield promoted by application of HYTb at 0.5
L/acre, applied with fungicide by foliar application. In a 60 acre test plot compared to a 26.48
acre control plot, the treated acreage gave about 12% increased yield compared to control.
Example 14
Treatment of Strawberry with Microbial Compositions
This example describes a representative method for obtaining increased strawberry crop
yield, using a microbial consortium. One skilled in the art will appreciate that methods that
deviate from these specific methods can also be used for increasing crop yield.
Treatment of strawberry with a microbial composition prepared similarly to A1006 plus
HYTb showed increases in final harvestable yield. All agronomic practices of fertilization,
cultivation, weed control, and pest control, were identical and side-by-side for both the treated
(Test) and control (Check) plots.
An increase in cumulative marketable production was promoted by application of
microbial composition and HYTb applied by drip irrigation. In these five independent trials, the
Sabrina variety was evaluated in the Huelva region of Spain. One week prior to plantlet
transplantation in the raised bed plots, 2L of the microbial composition plus 4L HYTb were
diluted in water and added to the drip irrigation per hectare, with the same application rate
performed at weeks 2, 4, and 6 post-planting. At weeks 3, 5, and 7, diluted microbial
composition was added at a rate of 1L/ha and diluted HYTb at a rate of 2L/ha. From week 9 to
the end of the harvest season, diluted microbial composition and HYTb were added at rates of
lL/ha each. In all five trials, the treatment boosted yield from 5% to 11% above parallel nontreated
plots, for an average of about an 8% yield increase across all five trials (FIG. 11).
Example 15
Treatment of Beetroot with Microbial Compositions
This example describes a representative method for obtaining increased beetroot crop
yield, using a microbial consortium. One skilled in the art will appreciate that methods that
deviate from these specific methods can also be used for increasing crop yield.
Treatment of beetroot with a microbial composition prepared similarly to A1006 plus
HYTb showed increases in final harvestable yield. All agronomic practices of fertilization,
cultivation, weed control, and pest control, were identical and side-by-side for both the treated
(Test) and control (Check) plots.
An increase in average harvested head weight was promoted by application of microbial
composition (2 L/acre) and HYTb (2 L/acre) applied by drip irrigation and HYTb ( 1 L/acre) by
foliar application. In an 8 acre test plot compared to a 9 acre control plot, the treated acreage
gave about 2.2-fold higher yield than control (FIG. 12).
Example 16
Treatment of Green Cabbage with Microbial Compositions
This example describes a representative method for obtaining increased green cabbage
crop yield, using a microbial consortium. One skilled in the art will appreciate that methods that
deviate from these specific methods can also be used for increasing crop yield.
Treatment of green cabbage with a microbial composition prepared similarly to Al 006,
or with HYTb, showed a strong increase in final harvestable yield. All agronomic practices of
fertilization, cultivation, weed control, and pest control, were identical and side-by-side for the
microbial composition- or HYTb-treated plots (Test) and control (Check) plots.
The trials showed an increase in cabbage yield promoted by application of microbial
composition (2 L/acre) and HYTb (2 L/acre) applied by drip irrigation and HYTb ( 1 L/acre) by
foliar application. Cabbages were harvested in two cycles, as represented by the "first cut"
harvest of cabbage heads and the later "second cut" of cabbage heads. As shown in FIG. 13A,
in a 10.9 acre test plot compared to a 14.9 acre control plot, the treated acreage gave about 18%
higher yield than control (first cut) and about 31% higher yield than control (second cut). As
shown in FIG. 13B, in a 3.7 acre test plot compared to a 1.5 acre control plot, the treated acreage
gave about 61% higher yield than control (first cut) and about 64% higher yield than control
(second cut).
Example 17
Seed and Tuber Treatment with HVId
This example describes a representative method for obtaining increased wheat and potato
crop yield using pre-treatment of the seed or seed tubers with HYTd. One skilled in the art will
appreciate that methods that deviate from these specific methods can also be used for increasing
crop yield.
Treatment of wheat seed or potato seed tubers prior to planting with HYTd prepared
using a microbial consortium similar to A1006 showed increases in final harvestable yield. All
agronomic practices of fertilization, cultivation, weed control, and pest control, were identical
and side-by-side for both the treated (Test) and control (Check) plots.
For wheat, seed was treated in a diluted suspension of HYTd, diluted at a rate of 3 mL of
HYTd in water per kg of seed. After coating seed and allowing air drying, treated seed was
planted and compared to identical plots of untreated seed. One acre parallel field plots showed
about 22% increase in wheat harvested yield (Table 3).
Potato seed treatment was performed by diluting HYTd in water and treating potato seed
at a rate of 1mL per kg of seed. After air drying, the treated potato seed was planted in parallel
with untreated control seed in 1200 meter, replicated plots. HYTd treated potato seed increased
potato yield 32% to 35% in two separate trials (Table 4).
Table 3. Yield from HYTd treated wheat seed
Table 4. Yield from HYTd treated potato seed
Example 18
Increased Stress Tolerance in Potato
This example describes a representative method for obtaining increased potato tuber
quality by treating with a microbial composition similar to Al 006 and HYTb during growth
under stressful field conditions.
Russet Burbank variety potato was grown under conventional conditions in a replicated
plot trial (four replicates) and either treated (microbial composition plus HYTb at 1 L each per
acre at planting, in furrow, followed by two foliar spray applications of HYTb at 1 L/acre at 55
days and again 85 days after planting) or untreated (control). Russet Burbank variety is prone to
lower quality under water, heat, or nutrient stress. In this trial, the microbial composition and
HYTb treatment enhanced tolerance to a stress-induced quality defect called hollow heart. Plots
treated with microbial composition had an incidence of 1.68% of harvested tubers with hollow
heart compared to the control with 8.35% hollow heart defects (Table 5).
Table 5. Potato hollow heart quality defects
* p<0.01 compared to untreated
Example 19
Cucumber Vigor Assay
Rapid plant-based functional assays can be used to quickly evaluate plant response to
new microbial compositions. Using a cucumber vigor and plant growth assay, this example
demonstrates that A1006 enhances the rate of plant leaf growth and expansion.
After pre-germination of cucumber seedlings in nutrient-soaked rolled germination paper
for four days, staged and synchronized plants were treated with a diluted mixture of liquid
fertilizer and microbial consortium. Plantlets were transplanted into prepared soilless growth
medium pre-treated with fertilizer and the tester solution. The microbial composition A1006
was diluted 1:2000 in a nutrient fertilizer media. As control treatment, an equivalent amount of
water added to nutrient media was compared. At least 18 plants of each treatment grown in
pots, including control plants, were randomized in flats, and grown under defined growth
conditions, controlling for temperature and light. After 18 days, the Leaf Area Index (LAI) of
the first true leaf of each plant was measured. The total plant wet weight was also recorded.
The data was analyzed by One-way ANOVA (Analysis Of Variance) and with post-hoc Tukey
test to compare samples within the experiment.
At day 18, the first leaf LAI rating promoted by A1006 treatment was significantly
greater than the control (FIG. 14).
In addition to, or as an alternative to the above, the following embodiments are
described:
Embodiment 1 is directed to a composition comprising the microbes in ATCC deposit
PTA-121755 (A1006).
Embodiment 2 is directed to a composition comprising five or more microbial species
selected from Bacillus spp., Pseudomonas spp., Lactobacillus spp., Desulfococcus spp.,
Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp., Ruminococcus spp.,
Leptospirillum spp., Halorhabdus spp., Clostridium spp., Xenococcus spp., Cytophaga spp., and
Candidatus spp.
Embodiment 3 is directed to a composition comprising ten or more microbial species
selected from Bacillus spp., Pseudomonas spp., Lactobacillus spp., Desulfococcus spp.,
Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp., Ruminococcus spp.,
Leptospirillum spp., Halorhabdus spp., Clostridium spp., Xenococcus spp., Cytophaga spp., and
Candidatus spp.
Embodiment 4 is directed to a composition comprising fifteen or more microbial species
selected from Bacillus spp., Pseudomonas spp., Lactobacillus spp., Desulfococcus spp.,
Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp., Ruminococcus spp.,
Leptospirillum spp., Halorhabdus spp., Clostridium spp., Xenococcus spp., Cytophaga spp., and
Candidatus spp.
Embodiment 5 is directed to a composition comprising each of Bacillus spp.,
Pseudomonas spp., Lactobacillus spp., Desulfococcus spp., Desulfotomaculum spp.,
Marinobacter spp., Nitrosopumilus spp., Ruminococcus spp., Leptospirillum spp., Halorhabdus
spp., Clostridium spp., Xenococcus spp., Cytophaga spp., and Candidatus spp.
Embodiment 6 is directed to a composition of any one of embodiments 2 to 5, wherein
the Bacillus spp. comprises one or more of Bacillus amyloliquefaciens, Bacillus pocheonensis,
and Bacillus clausii
Embodiment 7 is directed to a composition of any one of embodiments 2 to 6, further
comprising one or more of Microbacterium spp., Sporosarcina spp., Lysinibacillus spp.,
Nesterenkonia spp., Agrococcus spp., Acremonium spp., Sphingomonas spp., Micrococcus spp.,
Paenibacillus spp., Leucobacter spp., Brevundimonas spp., Rhizobium spp., Chitinophaga spp.,
Brevibacillus spp., Virgibacillus spp., Rummeliibacillus spp., Staphylococcus spp., or
Oceanobacillus spp.
Embodiment 8 is directed to a composition of embodiment 7, wherein the
Microbacterium spp. is Microbacterium testaceum, the Lysinibacillus spp. is Lysinibacillus
sphaericus, the Agrococcus spp. is Agrococcus terreus, and/or the Acremonium spp. is
Acremonium bacillisporum.
Embodiment 9 is directed to a composition of any one of embodiments 1 to 8, further
comprising one or more of chitin, chitosan, glucosamine, and amino acids.
Embodiment 10 is directed to a method comprising:
mixing a chitin-containing biological source with the composition of any one of
embodiments 1 to 9 to form a mixture;
fermenting the mixture; and
separating the fermented mixture into solid, aqueous, and lipid fractions.
Embodiment 11 is directed to the method of embodiment 10, wherein the chitincontaining
biological source comprises an aquatic animal or aquatic animal by-product, an
insect, or a fungus.
Embodiment 12 is directed to the method of embodiment 10, wherein the aquatic animal
is an aquatic arthropod.
Embodiment 13 is directed to the method of embodiment 12, wherein the aquatic
arthropod is shrimp, crab, or krill.
Embodiment 14 is directed to the aqueous fraction made by the method of any one of
embodiments 10 to 13.
Embodiment 15 is directed to the solid fraction made by the method of any one of
embodiments 10 to 13.
Embodiment 16 is directed to a method comprising contacting soil, plants, or plant parts
with the composition of any one of embodiments 1 to 9.
Embodiment 17 is directed to the method of embodiment 16, further comprising
contacting the soil, plants, or plant parts with one or more of chitin, chitosan, glucosamine, and
amino acids.
Embodiment 18 is directed to the method of embodiment 16 or 17, further comprising
contacting the soil, plants, or plant parts with the aqueous fraction of embodiment 14 or the solid
fraction of embodiment 15.
Embodiment 19 is directed to the method of any one of embodiments 16 to 18, further
comprising contacting the soil, plants, or plant parts with a liquid fertilizer.
In view of the many possible embodiments to which the principles of the disclosure may
be applied, it should be recognized that the illustrated embodiments are only examples and
should not be taken as limiting the scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our invention all that comes within the
scope and spirit of these claims.

We claim:
1. A composition comprising five or more microbial species selected from Bacillus spp.,
Pseudomonas spp., Lactobacillus spp., Desulfococcus spp., Desulfotomaculum spp.,
Marinobacter spp., Nitrosopumilus spp., Ruminococcus spp., Leptospirillum spp., Halorhabdus
spp., Clostridium spp., Xenococcus spp., Cytophaga spp., and Candidatus spp.
2. The composition of claim 1, comprising ten or more microbial species selected from
Bacillus spp., Pseudomonas spp., Lactobacillus spp., Desulfococcus spp., Desulfotomaculum
spp., Marinobacter spp., Nitrosopumilus spp., Ruminococcus spp., Leptospirillum spp.,
Halorhabdus spp., Clostridium spp., Xenococcus spp., Cytophaga spp., and Candidatus spp.
3. The composition of claim 2, comprising fifteen or more microbial species selected
from Bacillus spp., Pseudomonas spp., Lactobacillus spp., Desulfococcus spp.,
Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp., Ruminococcus spp.,
Leptospirillum spp., Halorhabdus spp., Clostridium spp., Xenococcus spp., Cytophaga spp., and
Candidatus spp.
4. The composition of claim 3, comprising each of Bacillus spp., Pseudomonas spp.,
Lactobacillus spp., Desulfococcus spp., Desulfotomaculum spp., Marinobacter spp.,
Nitrosopumilus spp., Ruminococcus spp., Leptospirillum spp., Halorhabdus spp., Clostridium
spp., Xenococcus spp., Cytophaga spp., and Candidatus spp.
5. The composition of any one of claims 1 to 4, wherein the Bacillus spp. comprises one
or more of Bacillus amyloliquefaciens, Bacillus pocheonensis, and Bacillus clausii.
6. The composition of any one of claims 1 to 5, further comprising one or more of
Microbacterium spp., Sporosarcina spp., Lysinibacillus spp., Nesterenkonia spp., Agrococcus
spp., Acremonium spp., Sphingomonas spp., Micrococcus spp., Paenibacillus spp., Leucobacter
spp., Brevundimonas spp., Rhizobium spp., Chitinophaga spp., Brevibacillus spp., Virgibacillus
spp., Rummeliibacillus spp., Staphylococcus spp., and Oceanobacillus spp.
7. The composition of claim 6, wherein the Microbacterium spp. is Microbacterium
testaceum, the Lysinibacillns spp. is Lysinibacillus sphaericus, the Agrococcus spp. is
Agrococcus terreus, and/or the Acremonium spp. is Acremonium bacillisporum.
8. The composition of any one of claims 1 to 7, further comprising one or more of
chitin, chitosan, glucosamine, and amino acids.
9. A method comprising:
mixing a chitin-containing biological source with the composition of any one of
claims 1 to 8 to form a mixture;
fermenting the mixture; and
separating the fermented mixture into solid, aqueous, and lipid fractions.
10. The method of claim 9, wherein the chitin-containing biological source comprises an
aquatic animal or aquatic animal by-product, an insect, or a fungus.
11. The method of claim 10, wherein the aquatic animal is an aquatic arthropod.
12. The method of claim 11, wherein the aquatic arthropod is shrimp, crab, or krill.
13. The aqueous fraction made by the method of any one of claims 9 to 12.
14. The solid fraction made by the method of any one of claims 9 to 12.
15. A method comprising contacting soil, plants, or plant parts with the composition of
any one of claims 1 to 8.
16. The method of claim 15, further comprising contacting the soil, plants, or plant parts
with one or more of chitin, chitosan, glucosamine, and amino acids.
17. The method of claim 15 or claim 16, further comprising contacting the soil, plants,
or plant parts with the aqueous fraction of claim 13 or the solid fraction of claim 14.
18. The method of any one of claims 15 to 17, further comprising contacting the soil,
plants, or plant parts with a liquid fertilizer.
19. The method of any one of claims 15 to 18, further comprising contacting the soil,
plants, or plant parts with one or more pesticides, one or more fungicides, one or more
herbicides, one or more insecticides, one or more plant hormones, one or more plant elicitors, or
combinations of two or more thereof.