SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 4, 2018, is named 0339-00003_SL.txt and is 14,802 bytes in size.

BACKGROUND OF THE INVENTION

Technical Field

The embodiments described herein relate to novel and unique methods, systems and processes for identifying, analyzing, monitoring, and controlling activities. Fermentation activities entail a wide range of endeavors directed toward agriculture, manufacturing, chemical processing.

The herein described process includes systems and methods for determining and characterizing the microbiome of a fermentation operation or setting, obtaining microbiome information, converting such information such that it is useful for controlling, enhancing, monitoring, detecting deviations, and predicting performance of the fermentation process.

Related Art

Fermentation is a process in which an agent causes transformation of a raw material into a finished product. During fermentation organic matter is decomposed in the absence or presence of air (oxygen) producing an accumulation of resulting fermentation product. Some of these products (for example, alcohol and lactic acid) are of importance to humans, and fermentation has therefore been used for their manufacture on an industrial scale.

Microorganisms like yeast, molds, and bacteria play an important role in the alcohol fermentation process for creating beer and wine, and the formation of acetic acid (vinegar). Lactic fermentation is driven by lactic-acid bacteria which break down monosaccharides into lactic acid. Lactic fermentation is used in the preparation of various sour milk products, yogurt, cheese, and bread. Many mold fungi (for example,Aspergillus niger) ferment sugar, resulting in the formation of citric acid. A large proportion of the citric acid used in the food-processing industry is obtained by microbiological means. Ethanol fuel is produced from the fermentation by yeast of common crops such as sugar cane, potato, cassava and corn to produce ethanol which is further processed to become fuel. The production of butyl alcohol and acetone industrially is important for the paint and lacquer industries. In the process of sewage treatment, sewage is digested by enzymes secreted by bacteria, to produce liquid and solid fertilizers, and biogas. Fungi have been employed to break down cellulosic wastes to increase protein content and improve in vitro digestibility. A wide variety of agroindustrial waste products can be fermented to use as food for animals, especially ruminants.

The processes described herein are useful for enhancing any fermentation process. The advantages of the herein described processes are shown for vinification, the process whereby fermentation changes grape juice into wine. However, it is understood that these methods can be applied for enhancement of other fermentation processes.

Winemaking or vinification, is the production of wine by fermentation of raw material, and for grape wine, that starts with the grapes. Factors affecting grape quality, known as the grape's terroir, include the variety of grapes, the weather during growing season, soil, time of harvest, and methods of pruning.

After harvesting the grapes, the fruit is crushed to produce juice, called must. The primary fermentation can be done with natural yeast normally already present on the grapes, visible as a powdery substance, or cultured yeast is added to the must. The sugar content of the grapes is monitored during fermentation and can be adjusted (by addition of sugar) since it affects both the taste and end product, as well as the speed of the fermentation.

During or after the primary fermentation, a secondary, or malolactic fermentation can be initiated by inoculation of desired bacteria which convert malic acid into lactic acid. This fermentation step can improve the taste of wine. During this secondary fermentation and aging process, fermentation continues very slowly in either stainless steel vessels or oak barrels.

Prior to bottling, the wine is usually filtered. Filtration results in clarification and microbial stabilization. In clarification, large particles that affect the visual appearance of the wine are removed. In microbial stabilization, the amount of yeast and bacteria are adjusted to prevent the likelihood of refermentation or spoilage.

As is evident from the winemaking steps described above, byproducts of fermentation by the microbial population or microbiome panel present in the soil, on the fruit, or during the winemaking process, contribute to the taste and quality of the wine.

Therefore, understanding the microbiome, and how it changes along each stage of vinification or wine production, would be advantageous and necessary for influencing the quality of the wine at every level. Using the herein described novel and unique sequencing methods, it is now possible to generate a unique identity for the wine, a genetic footprint, based on its microbiome. Such a footprint would allow winemakers to differentiate wines according to the microbiome panel, and detect and solve problems using bio-based controls such as Brettanomyces contamination, refermentation, mousiness, ropiness, mannitol, granium taint, diacetyl level, to name a few. These problems can be solved by bioremediation and/or changing the physical parameters, e.g. temperature, pH, enzymes, in the vinification process and influencing the microbiome community.

SUMMARY OF THE INVENTION

The present invention addresses the long-standing and unfulfilled need for better monitoring, analysis and control of fermentation activities, including, among others, those directed toward agriculture, biofuels, and food production.

The terms microbiome, microbiome information, microbiome data, microbiome population, microbiome panel and similar terms are used in the broadest possible sense, unless expressly stated otherwise, and would include: a census of currently present microorganisms, both living and nonliving, which may have been present months, years, millennia or longer; a census of components of the microbiome other that bacteria and archea, e.g. viruses and microbial eukaryotes; population studies and characterizations of microorganisms, genetic material, and biologic material; a census of any detectable biological material; and information that is derived or ascertained from genetic material, biomolecular makeup, fragments of genetic material, DNA, RNA, protein, carbohydrate, metabolite profile, fragment of biological materials and combinations and variations of these.

As used herein, the terms real-time microbiome data or information includes microbiome information that is collected or obtained at a particular setting during the fermentation process, for example soil, plant/fruit samples taken during a planting or harvesting, must, sampling of wine during alcoholic fementation (beginning, middle and end, or depending on parameters such as alcoholic graduation, amount of sugar, density), sampling during malolactic fermentation (beginning, middle and end, or depending on amount of malic and acetic acid), barrel (beginning, middle and end, or months) and bottling.

As used herein, the terms derived microbiome information and derived microbiome data are to be given their broadest possible meaning, unless specified otherwise, and includes any real-time, microbiome information that has been computationally linked or used to create a relationship such as for example evaluating the microbiome of milk before, during, and after fermentation, or evaluating the microbiome between planting and harvesting of grapes. Thus, derived microbiome information provides information about the fermentation process setting or activity that may not be readily ascertained from non-derived information.

As used herein, the terms predictive microbiome information and predictive microbiome data are to be given their broadest possible meaning, unless specified otherwise, and includes information that is based upon combinations and computational links or processing of historic, predictive, real-time, and derived microbiome information, data, and combinations, variations and derivatives of these, which information predicts, forecasts, directs, or anticipates a future occurrence, event, state, or condition in the industrial setting, or allows interpretation of a current or past occurrence. Thus, by way of example, predictive microbiome information would include: a determination and comparison of real-time microbiome information and the derived microbiome information of quality of wine, i.e. abundance of a specific microorganism in a sample and possible positive or negative effect on the fermentation process; a comparison of real-time microbiome information collected during the fermentation of cheese and the quality of cheese.

Real time, derived, and predicted data can be collected and stored, and thus, become historic data for ongoing or future decision-making for a process, setting, or application.

In one embodiment of the invention is provided a method of classifying a microorganism, comprising: obtaining a nucleic acid sequence of a 16S ribosomal subunit, an ITS, internal transcribed spacer, and optionally, a single copy marker gene, of a first microbe; and comparing said nucleic acid sequence of a first microbe to a reference; and identifying the first microbe at the strain level or sub-strain level based on the comparing.

In another embodiment is provided a novel method of profiling a microbiome in a sample, comprising: obtaining nucleic acids sequences of a 16S ribosomal subunit, an ITS, and a marker gene, from at least one microorganism in a sample; analyzing said at least one microorganism within said sample based upon the nucleic acids sequences obtained; and determining a profile of the microbiome based on said analyzing. Using 16S rDNA in combination with another single-copy marker gene provides prokaryotic species boundaries at higher resolution and allows identification of microbial diversity at the strain level. The novelty of this method is in the fact that unlike what is currently taught and used in the art, instead of combining the measurement of 16S region with a functional gene as is taught in the art, we combine the 16S region with single-copy marker genes (described in Sunagawa et al., 2013, Nature Methods 10, 1196-1199). This methodology required sequencing all the DNA in a sample in order to get a high filogenetic resolution level. The method described herein, reduces the amount of sequencing data needed to identify species at high filogenetic resolution because the 16S amplicons and the single-copy marker genes produce an alignment rate below 7% and a false discovery rate below 10%.

In another embodiment is provided a novel method for sequencing two libraries in one sequencing run, by pooling the prepared 16S and ITS libraries, and providing appropriate primers for sequencing both 16S and ITS in a sequencing method.

In some embodiments, determining a profile of the microbiome in said sample can be based on 50 or fewer microbes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewer microbes, 100 or fewer microbes, 200 or fewer microbes, 300 or fewer microbes, 400 or fewer microbe, 500 or fewer microbes, 600 or fewer microbes, 700 or fewer microbes, or 800 or fewer microbes. In some embodiments determining a profile of the microbiome in said sample has an accuracy greater than 70% based on the measurements. In some embodiments, analyzing uses long read sequencing platforms.

In yet another embodiment is provided a process including: analyzing a material from a location associated with a fermentation process; obtaining microbiome information, selected from real time microbiome information, derived microbiome information and predictive microbiome information; and performing an evaluation on the microbiome information, the evaluation including: a relationship based processing including a related genetic material component and a fermentation setting component; and a bioinformatics stage; whereby the evaluation provides information to direct the fermentation process.

In a further embodiment is provided operations and methods having one or more of the following features: wherein the real time microbiome information is selected from material selected from the group consisting of soil samples, soil sample taken during a planting, soil sample taken during growth, soil sample taken during harvesting, fermentation sample taken at the beginning of a fermentation process, in the middle of a fermentation process, at the end of a fermentation process, any time during a fermentation process; wherein the bioinformatics stage has one or more of the following: submitting the raw DNA sequencing data to bioinformatics pipeline for performing microbiome analysis, including demultiplexing and quality filtering, OTU picking, taxonomic assignment, phylogenetic reconstruction, compiling metadata, diversity analysis, and visualization.

Still in another embodiment is provided a method of controlling a fermentation operation including: analyzing a material from a location associated with an fermentation operation to provide a first microbiome information; associating the first microbiome information with a condition of the operation; obtaining a second microbiome information; associating the second microbiome information with the first microbiome information; and, evaluating the first microbiome information, the associated condition, and the second microbiome information, the evaluation including bioinformatics pipeline for performing microbiome analysis including demultiplexing and quality filtering, OTU picking, taxonomic assignment, plytogenetic reconstruction, compiling metadata, diversity analysis, and visualization; whereby the evaluation identifies a characteristic of the operation; and, directing the fermentation operation based in part on the identified characteristic of operation; whereby the fermentation operation is based upon the evaluation of microbiome information.

Yet still in another embodiment is provided a method for directing a fermentation operation including: analyzing a sample from a location associated with a fermentation operation; obtaining microbiome information; and, performing an evaluation on the microbiome information, whereby the evaluation provides information to direct the fermentation operation.

In another embodiment is provided operations and methods having one or more of the following features: wherein, the microbiome information has real time microbiome information; wherein, the microbiome information has derived microbiome information; wherein, the microbiome information has predictive microbiome information; wherein the analysis has selection and sequencing of the material; wherein the analysis has extracting genetic material from the material; wherein the analysis has preparation of libraries; wherein the analysis has extracting material including genetic material selected from the group consisting of a rRNA gene 16S, Internal transcribed spacer (ITS); wherein the analysis has providing a phylogenetic tree; wherein the analysis has a correction step; wherein the analysis has an extraction procedure selected from the group consisting of beating, sonicating, freezing and thawing, and chemical disruption; wherein the analysis has amplification of at least a portion of the material; wherein the analysis has providing a genetic barcode to a sample of the material; wherein the microbiome information defines a phylogenetic tree; wherein the microbiome information has a OTU; wherein the microbiome information defines an OTU; wherein the microbiome information defines a biogeographical pattern; wherein the microbiome information has information obtained from the 16S rRNA and another marker gene; wherein the another marker gene is metal-dependent proteases with possible chaperone activity; wherein the evaluation has forming an n-dimensional plot, where n is selected from the group of integers consisting of 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, and 14; wherein the evaluation has measuring a change in gene sequences; wherein the evaluation has measuring a change in gene sequences and using the measured change as a molecular clock in the evaluation to determine the related nature of material; and wherein the material is selected from the group consisting of soil, agricultural material, material from dairy processing, a material from a fermentation operation.

There is further provided systems, operations and methods having one or more of the following features: wherein at least a portion of the information resulting from the evaluation is displayed in a two dimensional plot; wherein at least a portion of the information resulting from the evaluation is displayed in a three dimensional plot; wherein at least a portion of the information resulting from the evaluation is displayed in a plot including colors associated with microbiome information; wherein at least a portion of the information resulting from the evaluation is displayed in a plot including colors associated with a type of information selected from the group consisting of microbiome information and non-genetic information; each type of information including a different color; wherein at least a portion of the information resulting from the evaluation is displayed in a plot including colors associated with a type of information selected from the group consisting of microbiome information and non-genetic information; each type of information including a different color; and the non-genetic information selected from the group consisting of temperature, geographical location, climate; wherein at least a portion of the information resulting from the evaluation is transmitted to a memory storage device; wherein at least a portion of the information resulting from the evaluation is communicated to a controller; wherein at least a portion of the information resulting from the evaluation is displayed in a two dimensional plot; and, wherein at least a portion of the information resulting from the evaluation is displayed in a three dimensional plot. In some embodiments, the system can further comprise a user interface configured to communicate or display a report to a user.

In one aspect, the methods of the invention allow the identification of microorganisms capable of imparting one or more beneficial property to one or more phases of a fermentation process. The variability in the microbial populations present in the sample can be used to support a directed process of selection of one or more microorganisms for use in a phase of a fermentation process and for identifying particular combinations and abundances of microorganisms which are of benefit for a particular purpose, and which may never have been recognized using conventional techniques.

The methods of the invention may be used as a part of a plant breeding program. The methods may allow for, or at least assist with, the selection of plants which have a particular genotype/phenotype which is influenced by the microbial flora, in addition to identifying microorganisms and/or compositions that are capable of imparting one or more property to one or more plants.

In one aspect the invention relates to a method for the selection of one or more microorganism(s) which are capable of imparting one or more beneficial property to a plant to be used as raw material in a fermentation process. In other words, the process will allow for enrichment of suitable microorganisms within the plant microbiome. Such microorganism(s) may be contained within a plant, on a plant, and/or within the plant's growing soil or water. It should be appreciated that as referred to herein a “beneficial property to a plant” should be interpreted broadly to mean any property which is beneficial for any particular purpose including properties which may be beneficial to human beings, other animals, the environment, a habitat, an ecosystem, the economy, of commercial benefit, or of any other benefit to any entity or system. Accordingly, the term should be taken to include properties which may suppress, decrease or block one or more characteristic of a plant, including suppressing, decreasing or inhibiting the growth or growth rate of a plant. The invention may be described herein, by way of example only, in terms of identifying positive benefits to one or more plants or improving plants. However, it should be appreciated that the invention is equally applicable to identifying negative benefits that can be conferred to plants.

Such beneficial properties include, but are not limited to, for example: improved growth, health and/or survival characteristics, suitability or quality of the plant for a particular purpose, structure, color, chemical composition or profile, taste, smell, improved quality. In other embodiments, beneficial properties include, but are not limited to, for example; decreasing, suppressing or inhibiting the growth of a plant; constraining the height and width of a plant to a desirable size; regulate production of and/or response to plant pheromones (resulting in increased tannin production in surrounding plant community and decreased appeal to foraging species).

As used herein, “improved” should be taken broadly to encompass improvement of a characteristic of a plant or a fermentation process which may already exist in a plant or process prior to application of the invention, or the presence of a characteristic which did not exist in a plant or process prior to application of the invention. By way of example, “improved” growth should be taken to include growth of a plant where the plant was not previously known to grow under the relevant conditions.

As used herein, “inhibiting and suppressing” and like terms should be taken broadly and should not be construed to require complete inhibition or suppression, although this may be desired in some embodiments.

The term “microbes”, “microorganisms” as used herein should be taken broadly. It refers to any single-celled organisms, bacteria, archaea, protozoa, and unicellular fungi and protists. By way of example, the microorganisms may include Proteobacteria (such asPseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, SinorhizobiumandHalomonas), Firmicutes (such asBacillus, Paenibacillus, Lactobacillus, Mycoplasma, andAcetobacterium), Actinobacteria (such asStreptomyces, Rhodococcus, Microbacterium, andCurtobacterium), and the fungi Ascomycota (such asTrichoderma, Ampelomyces, Coniothyrium, Paecoelomyces, Penicillium, Cladosporium, Hypocrea, Beauveria, Metarhizium, Verticullium, Cordyceps, Pichea, andCandida, Basidiomycota (such asCoprinus, Corticium, andAgaricus) and Oomycota (such asPythium, Mucor, andMortierella).

In yet another embodiment, the present disclosure provides a method for detecting contamination in a fermentation sample, comprising determining the microbiome from a fermentation sample, wherein the method comprises detecting at least one marker of a microorganism and preferably two markers of a microorganism; and a computer system for determining a microbiome profile in a sample, the computer system comprising: a memory unit for receiving data comprising measurement of a microbiome panel from a sample; computer-executable instructions for analyzing the measurement data according to a method of described herein; and computer-executable instructions for determining potential microbial contamination in the sample or fermentation process based upon said analyzing. In some embodiments, the computer system further comprises computer-executable instructions to generate a report of the presence or absence of the at least one contamination microorganism in the sample. In some embodiments, computer system can further comprises a user interface configured to communicate or display said report to a user.

The present disclosure provides a computer readable medium comprising: computer-executable instructions for analyzing data comprising measurement of a microbiome profile from a fermentation sample obtained from a fermentation process or environment, wherein the microbiome profile comprises at least one marker and preferably two markers selected from at least one microbe; and computer-executable instructions for determining a presence or absence of a contamination in the fermentation process based upon the analyzing.

Examples of machine learning algorithms that can be used include, but are not limited to: elastic networks, random forests, support vector machines, and logistic regression. The algorithms provided herein can aid in selection of important microbes and transform the underlying measurements into a score or probability relating to, for example, grape quality, wine quality, presence or absence of contamination, treatment response, and/or classification of organic soil status.

The present disclosure provides a kit, comprising: one or more compositions for use in measuring a microbiome profile in a fermentation sample obtained from fermentation process or environment thereof, wherein the microbiome profile comprises at least one marker and preferably two markers to at least one microbe; and instructions for performing any of the preceding methods. In some embodiments, a kit can further comprises a computer readable medium.

Kit reagents may in one embodiment comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of a microorganism. In another embodiment, the kit comprises at least one pair of oligonucleotides that hybridizes to opposite strands of a genomic segment of a microorganism, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the 16S, ITS, and/or marker gene of the organism present in the sample. In one embodiment, the oligonucleotide is completely complementary to the genome of the individual. In another embodiment, the kit further contains buffer and enzyme for amplifying said segment. In another embodiment, the reagents further comprise a label for detecting said fragment.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

