Success cases. blight Bitter rot Black rot Botrytis bunch rot Cercospora

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1 Esca/Petri disease Esca Complex Petri disease Botryosphaeria dieback Black foot disease Eutypa dieback Phytophthora crown and root rot Armillaria root rot Angular leaf scorch / Rotbrenner Aspergillus rot Cladosporium rot Crown gall tumors Ripe rot Alternaria rot Angular leaf spot Anthracnose Bacterial blight Bitter rot Black rot Botrytis bunch rot Cercospora leaf spot / Cladosporium leaf spot Dematophora root rot Downy mildew Leaf rust Macrophoma rot Phomopsis dieback Phymatotrichum root rot (Texas) Pierce's disease Powdery mildew Septoria leaf spot Tar spot Verticillium wilt White rot Zonate leaf spot Esca/Petri disease Esca Complex Petri Success cases disease Botryosphaeria dieback Black foot disease Eutypa dieback Phytophthora crown and root rot Armillaria root rot Angular leaf scorch / Rotbrenner Aspergillus rot Cladosporium rot Crown gall tumors Ripe rot Alternaria rot Angular leaf spot Anthracnose Bacterial blight Bitter rot Black rot Botrytis bunch rot Cercospora leaf spot / Cladosporium leaf spot Dematophora root rot Downy mildew Leaf rust Macrophoma rot Phomopsis dieback Phymatotrichum root rot (Texas) Pierce's disease Powdery mildew Septoria leaf spot Tar spot Verticillium wilt White rot Zonate leaf spot Esca/Petri disease Esca Complex Petri disease Botryosphaeria dieback Black foot disease Eutypa dieback Phytophthora crown and root rot Armillaria root rot Angular leaf scorch / Rotbrenner Aspergillus rot Cladosporium rot Crown gall tumors Ripe rot Alternaria rot Angular leaf spot Anthracnose Bacterial blight Bitter rot Black rot Botrytis bunch rot Cercospora leaf spot / Cladosporium leaf spot Dematophora root rot Downy mildew Leaf rust Macrophoma rot Phomopsis dieback Phymatotrichum root rot (Texas) Pierce's disease Powdery mildew Septoria leaf spot Tar spot Verticillium wilt White rot Zonate leaf spot C R E A T O R S

2 preface In microbiology, just like in every aspect of life, the essential is invisible to the eye. From the management of Biome Makers, we are proud of developing WineSeq, a technology solution unheard in wine industry. From characterization to interpretation of all the microbial communities that affect grape s quality, through those responsible for the fermentative process. Like explorers of a new universe, the twenty experts that constitute Biome Makers today in different areas of knowledge microbiology, genetics, informatics, are feeling very lucky for discovering and characterizing new bacteria and yeasts, that turn wine s existence into a true work of art, a great experience through a symphony of aroma and flavor. With this document we want to share with you a compilation of our work in We have selected several success cases where the greatest satisfaction has been helping people that share our passion for enology. Alberto Acedo Adrián Ferrero Biome Makers founders

3 D E S C R I P T I O N Success cases WineSeq, our nextgeneration microbiome soil monitoring test, has been applied to more than 3000 samples in 18 countries. So we want to show you some of our success cases that have guaranteed that we are able to support winemakers to better understand the vine and wine diseases and the risks and enhancers in their fermentative processes.

4 index 4 12 Viticulture cases Fermentation cases Differentiation cases Bioremediation cases

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6 1 VITICULTURE CASES Controlling vine diseases, preparing soil, fertilizing and monitoring grape development are not easy tasks. Microbiology helps the winemaker to better identify all the elements of viticulture processes that could be unknown if WineSeq is not applied. CASE 1 If you think as a Trichoderma you could be a biocontrol agent of Black Foot CASE 2 How much do you know about Eutypa dieback? When we talk about Pedro Ximénez, we CASE 3 think of sweet- CASE 4 ness, but the wood disease has a bitter taste Please, allow me to introduce myself: I m Black foot disease CASE 5 If you are going to plant a vineyard, make sure you buy healthy plants CASE 6 Do you want to save money on your vineyard? Analyze soil and vines! 5

7 If you think as a Trichoderma you could be a biocontrol agent of Black Foot A high production and internationally recognized winery that produces wines from Tempranillo in Ribera del Duero. Vineyard on a slope and limestone soil. The vines from the bottom of the slope and near the river showed a good appearance and an optimal yield. However, in the upper part, successive replantings were used since the vines experience recurrently low growth, low development and yield. Microbiology of the case PATHOGEN: Campylocarpon fasciculare SEVERITY 90 out of 100 A WineSeq test was carried out in the soil and the plant to detect the microbial community in the vineyard. The results showed the presence of pathogens associated with Black Foot disease in both the lower part and upper parts of the plot. However, we also detected a high abundance of Trichoderma in the healthy vines, corresponding to the area close to the river, which seem to be acting as biocontrol agents, stopping the development of the pathogenic fungus causing the Black Foot. These species were not found in the sections of the vineyards with health problems, so the development of the disease in the vines of the upper side of the plot spread unchecked. Once the effectiveness of Trichoderma on the pathogen was verifiedwith WineSeq tools, we recommended the application of a biological treatment from the biocontrol agent and the winery recovered the 80% yield of the vineyard. 6

8 How much do you know about Eutypa dieback? A small production winery that produces highvalue biodynamic wines from Cabernet Sauvignon in Napa Valley. Shortages in vegetative development and the presence of rachitic bunches. The leaves were smaller than usual and appeared chlorotic. The shoots did not develop normally and the internodes were shortened. Gradually, dark spots were detected on the wood as well as incomplete flowering, or millerandage. The problem resulted in a decrease in the yield, the qualitative aspect of the vineyard and the deterioration of the vine. Microbiology of the case PATHOGEN: Eutypa lata SEVERITY 100 out of 100 We performed a WineSeq test using the vineyard soil to detect the active microbial community. The results showed the presence of pathogens associated with the fungal disease of the vine wood called Eutypa dieback with a degree of 100 out of 100 severity. From the decisive identification of the pathogen and its degree of incidence, we implemented a series of measures, such us changes in work habits and preventive practices to limit the development of the fungus and restore the healthy status of the plants. We planned an analytical review in six and twelve months. Among these actions, we recommended a late and short pruning, the quantification of the damaged vines, as well as removing the infected wood from the vineyard soil after pruning. 7

9 When we talk about Pedro Ximénez, we think of sweetness, but the wood disease has a bitter taste A small production winery that makes sweet wines from the Pedro Ximénez variety. The vineyard where Pedro Ximénez is grown is planted on clay and limestone soil. The plot presented with several areas where the leaves did not experience adequate development. Dark spots in the wood and an incomplete flowering, or millerandage, were detected. Symptoms of mildew and Gray rot in the affected zones also appeared. In some areas, replanting was chosen as a palliative measure. Microbiology of the case PATHOGEN: Eutypa lata SEVERITY 100 out of 100 The vineyard was divided into plots according to the symptoms detected in the vines and the vines age. A WineSeq test was carried out in the soil of each plot to identify the microbial community present. The results showed the presence of pathogens associated with the fungal disease of the vine wood Eutypa dieback. The degree of severity was 100% and included a complex of pathogens associated with Yesca, Plasmopara viticola and Botrytis cinerea. demonstrates the effectiveness of the WineSeq testing in the differentiation of pathogens associated with wood diseases. Based on the analysis obtained from WineSeq reports, the winery knew to plant a new vineyard saved the cost of replanting vines. 8

10 Please, allow me to introduce myself: I m Black foot disease A small production and high-value winery that makes wines from selected plots in Napa Valley. The WineSeq team studied the drone images of the winery and serious problems were detected in different locations of the vineyard. We visited the plot to evaluate the visual aspect of the vines. They showed anomalous growth and the manager of the winery informed us that the yield had diminished over the last two harvests. Microbiology of the case PATHOGEN: Dactylonectria vitis SEVERITY 80 out of 100 The vineyard was divided into subplots according to the results obtained from the drone analysis. A WineSeq test was performed in the soil of each plot to detect the microbial community present in the vineyard. The results showed the presence of pathogens associated with Black Foot disease. The disease was present in the plots of the western area and a correlation was established with the data obtained by the drone. We established a biocontrol plan and the application of Trichoderma. Through the combination of drone image analysis, WineSeq testing and customized recommendations, the winery recovered the damaged plots and only lost 20% of the production that vintage. 9

11 If you are going to plant a vineyard, make sure you buy healthy plants A vine-grower with a forty-five acre vineyard in California wants to plant twelve new acres of Chardonnay. The vine-grower purchased vines from a nursery at his local market. He wanted to confirm the absence of pathogens in the acquired plant material prior to developing the new Chardonnay new vineyard. The vine-grower selected two different rootstocks. We implement a protocol from the WineSeq test to determine the microbial population of the plant lots defined. Starting with plant material packed in bundles of 25 vines located in a cool and humid place, we selected five plants from different bundles arbitrarily for each combination of rootstock. Microbiology of the case PATHOGEN: Phomopsis viticola SEVERITY 70 out of 100 A WineSeq test was conducted with the sample plant material to detect the active microbial community. The results showed the presence of pathogens associated with diseases of wood and root at 27%. The pathogenic fungi found corresponded to Petri disease, Phomopsis viticola and root diseases such as Cylindricarpon spp. This illustrates the importance of microbiological analysis of plant material prior to planting a vineyard. Hefty economic losses can result if an assessment of the sanitary status of the plants is not made. In this case, WineSeq successfully diagnosed pathogens prior to planting and a loss of $100,000 was avoided. 10

12 Do you want to save money on your vineyard? Analyze soil and vines! A selection of 25 vineyard plots located in the United States and Spain where Pierce s disease thrives. We proposed a protocol to better understand the properties and behaviour of Xylella fastidiosa, which is responsible for the development of Pierce s Disease. WineSeq also investigated the vectors that influence Pierce s spread in order to assess impact and contribute to the search for a viable solution that limits its expansion. Microbiology of the case PATHOGEN: Xylella fastidiosa SEVERITY 80 out of 100 We administered a WineSeq test on vineyard soils and plants to detect the active microbial community. We found several Xylella fastidiosa focal points in five of the analyzed plots. The pathogen was in both the plant and the soil of the lower areas of the plots closest to the rivers. It is not usual to find this bacteria in soil since it is propagated by insects, mainly hemipteria of the subfamily Cicadellinae and the family Cercopidae. We implemented measures to limit the fungus development although there are currently no known methods of curative control. The focal points were located in the plots and WineSeq evaluation indicated isolating these areas as well as minimizing the accumulation of plant material from the perimeter. We also recommended the use of resistant clones and plants certified free of pathogens to further protect the affected vineyards. If you want to save money on your vineyard do not hesitate to analyze the soil and vines. 11

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14 2 FERMENTATION CASES CASE 1 It is usual that many winemakers and enologists experience problems in the fermentation process or perhaps they have doubts about certain spoilage yeasts that modify the organoleptic profile of their wines. WineSeq has been able to respond to many of the issues affecting fermentation processes. CASE 3 CASE 5 Love me twice even on a frozen planet Dance me to the end of Brett Call me glabatra, Candida glabrata CASE 2 CASE 4 CASO 6 How to manage fermentation kinetics How to find Hanseniaspora valbyensis during an advanced fermentative process Shall we uncork a bottled wine lot? CASE 7 Don t stop me now CASE 8 Do you feel the power of the microbiome? CASE 9 Would you leave fermentation to a machine alone? CASE 10 Solutions for abnormal fermentation in the presence of Brett CASE11 Lactic bacteria sometimes plays tricks CASE 12 Biogenic amines strike again; high ph is their greatest partner CASE 13 Don t blame everything on Brett 13

15 Love me twice even on a frozen planet A low-production and internationally recognized winery that makes organic wines and practices sustainable viticulture. Signs of re-fermentation after refrigeration during the elaboration of a sweet wine, increase in volatile acidity and the alteration of the product s taste. Microbiology of the case Problematic yeast: Saccharomyces kudriavzevii Saccharomyces cerevisiae YEAST CONCENTRATION 3, cells/ml 30,9% Sachharomyces kudriavzevii Non-Saccharomyces yeasts We performed WineSeq tests on the must and the fermentative processes. The results detected a cryophilic yeast, which started to dominate the population at the lower temperature, preventing the stoppage of the process and giving rise to a re-fermentation. of the yeast population showed us the displacement that Saccharomyces cerevisiae suffered from the cryophilic yeast Saccharomyces kudriavzevii, after the application of a cold process during the tenth day of fermentation. WineSeq suggested the inoculation of a cryotolerant yeast such as Saccharomyces to avoid contaminatio during fermentative maceration, as well as a microbiological treatment of the deposits where cryophilic fermentative yeast species are detected by the addition of sulfur dioxide. Another option was to discard the affected tanks for the winemaking process. The winemaking process was saved and the wine produced was able to arrive to market. 14

16 How to manage fermentation kinetics A low-production and internationally recognized winery that produces organic wines and prcatices sustainable viticulture. High levels of lactic acid in the Pied de Cuve. Values of 2 g/l of L-lactic acid were obtained at g/l must density and without any L-malic acid consumption. Microbiology of the case Problematic yeast: Lachancea thermotolerans Other yeasts YEAST CONCENTRATION 8, cells/ml 24,6% Lanchacea thermotolerans Saccharomyces yeasts A WineSeq test was administered during the beginning of the alcoholic fermentation in the Pied de Cuve, arranged as activators of the fermentation process. The non-saccharomyces yeast, Lanchancea thermotolerans, was identified. This species is related to the release of L-lactic acid from the consumption of sugars during fermentation, as well as high levels of glycerol. Palliative measures were recommended to increase the sulfur content and the fermentation temperature slightly to favour the development of indigenous Saccharomyces cerevisiae or the inoculation of selected yeasts. The winery carried out these actions and solved the fermentation processes, avoiding an excessive increase in the wine total acidity. The winemaking process was successful and the winery was able to sell the wine with the help of WineSeq. 15

17 Dance me to the end of Brett Spontaneous fermentation from selected plots of Cabernet sauvignon in an ecological winery. The wine had a recurrent organoleptic problem: a high content of 4-ethylphenol (which corresponds to the aromatic descriptor of horse sweat). This resulted in a fall in quality, a complete loss in the market and, as a consequence, an economic depreciation of the wine. Microbiology of the case Problematic yeast: Brettanomyces bruxellensis Saccharomyces yeasts YEAST CONCENTRATION 1, cells/ml 14,7% Brettanomyces bruxellensis Other yeasts WineSeq detected high levels of Brettanomyces bruxellensis during the fermentation process when we launched the WineSeq test. The preferred option would be an increase in the sulfur content which would stop in some cases the development of Brettanomyces, but this practice clashed with the winery s philosophy: to reach a constant reduction of sulfur level over the years. When the microbiology of the soils and grapes was analyzed, WineSeq detected Brettanomyces bruxellensis in all the samples. Understanding and respecting the viticulture practices of the winery was important to solve the case. The winery produced a fertilizer from fermentation lees that was contaminated with Brettanomyces from the cellar. Thereby, the soil was inoculated with this yeast and it was later transmitted to the grapes and to the cellar, thus feeding back the contamination. Once the problem was identified, WineSeq recommended completely halting use of the biofertilizer. It was possible to eliminate the microbiological origin of the volatile phenols production in the winery and stabilize the wine value in the market. The winery saved itself $35,

18 How to find Hanseniaspora valbyensis during an advanced fermentation process A small production and high-value winery that makes unique wines from a plot in Napa Valley. A variation in the fermentation kinetics at the beginning of the spontaneous fermentation of a must located in a five thousand liter stainless steel tanks was detected by WineSeq. Tests also found acetic acid and an increase in acidity volatility to higher levels than initially parameterized. It was discovered high levels of acetate 2-phenylethyl, a volatile that modifies wines organoleptic characteristics adding a smell like roses or honey and altering the original sensorial profile. Microbiology of the case Problematic yeast: Hanseniaspora valbyensis YEAST CONCENTRATION 4, C/mL Saccharomyces yeasts 24,6% Hanseniaspora valbyensis Non-Saccharomyces yeasts We implemented the WineSeq tests from the soil to the plant, the must and the fermentation process. A remarkable presence of Hanseniaspora valbyensis was detected. This is an apiculated yeast whose presence is usually restricted to the first fermentative stages and whose impact on the sensory properties of wines is scarce. However, the Hanseniaspora strain detected withstood the fermentative conditions and participated in the later stages of the fermentation. This yeast was not detected until the corresponding analysis was performed in the winery. With WineSeq testing, we could determine the origin of the anomaly during the winemaking process and stop its possible effect on the winery's machinery and facilities. High concentrations of these yeast species in the early stages of fermentation allowed us to anticipate their negative effect on the wine s taste. Thus, the WineSeq test in the must helped the winery make an appropriate decision about making spontaneous or inoculated fermentations with selected yeasts. 17

19 Call me glabatra, Candida glabatra A small-production and high-value winery that makes wines according to a sustainable model in different geographical areas. WineSeq detected frequent stops in the beginning of the alcoholic fermentation of a Sauvignon blanc must. Also, there was an increase in the levels of acetic acid that did not seem to come from the acetic bacteria according to classical microbiological analysis. Microbiology of the case Problematic yeast: Candida glabrata Saccharomyces yeasts YEAST CONCENTRATION 4, cells/ml 37,7% Candida glabrata Non-Saccharomyces yeasts We implemented the WineSeq test to identify the microbial populations from the plot soil, the vines, the must and the beginning of the spontaneous fermentation in the cellar. Testing detected an important concentration of Candida glabatra and Rhodotorula yeasts. We performed an implantation study on the fermented process and the in-vitro test evidenced the presence of non-saccharomyces and Candida glabrata as a species widely represented in the must. This discovery was associated with the increase in volatile acidity. Its origin was determined to be due to human or environmental contamination. WineSeq recommended the controlled addiction of sulfur dioxide and filtration of a selected Saccharomyces bayanus strain to quickly conclude fermentative processes, implantation and save production. 18

20 Shall we uncork a bottled wine lot? A high production and internationally recognized winery that produces wines in different Appellations of Origin. 200 bottled red wine cases presented organoleptic anomalies as herbaceous scents not typical of the winery. Initially it seemed to be an issue of contamination of the wine lot. Microbiology of the case Problematic yeast: Aureobasidium pullulans YEAST CONCENTRATION 3, C/mL Saccharomyces yeasts 14,8% Aureobasidium pullulans Non-Saccharomyces yeasts We implemented a WineSeq test protocol of the grape bunches, the soil and the wine. The yeast-fungus Aureobasidium pullulans was detected in the grapes at higher levels and its presence in the wine samples analyzed was surprisingly high. Normally, this fungus is found in the grapes skin but disappears quickly after pressing and the beginning of the fermentation process. This fungus was responsible for the production of high concentrations of varietal terpenes (aromatic descriptors of the herbaceous family), through the liberation of glycolytic enzymes. Likewise, the fungus was detected in the oak barrels where the wine was aged. 19

21 Don t stop me now A small production and high-value winery that produces wines according to a sustainable model in different geographical areas. The winery identified a premature stop of the fermentation of a Tempranillo must at 10% ethanol concentration. Microbiology of the case Problematic yeast: Pichia membranifaciens Saccharomyces yeasts YEAST CONCENTRATION 6, cells/ml 22,8% Pichia membranifaciens Non-Saccharomyces yeasts We developed a WineSeq test of the fermentation process and detected a high level of Pichia membranifaciens. Its presence in must-wine is considered a contaminant, due to its capacity to produce killer toxins, which can hinder the development of the fermentation process. Also, due to its oxidative metabolism, it was able to release negative compounds for wine quality such as acetaldehyde, and even form a film on the surface of the wine in the tank. The inoculation of Saccharomyces bayanus and the contribution of organic nutrients to encourage the reactivation of fermentation was recommended. With WineSeq s recommendations, the winery was able to finish the winemaking process and saved $18,

22 Do you feel the power of the microbiome? A small production and high-value group of wineries that make wines from selected plots in Napa Valley. The main objective of this project was the characterization of the microbiome (fungi/yeasts and bacteria) associated with different critical points in the winemaking process of three wineries. We are also defining the relationship and impact of the resident microbiology in the winery (surfaces and machinery) and fermentative processes. A WineSeq test protocol and timing were established during three key moments along the winemaking process: before harvesting, during harvesting and after harvesting. The control points are also marked in the hoppers, destemmer-crusher, pipes and pumps and fermentation tanks. Saccharomyces yeasts Microbiology of the case Problematic yeast: Metschnikowia YEAST CONCENTRATION 2, Cels/mL 16,4% Metschnikowia Non-Saccharomyces yeasts The WineSeq test prior to the harvest gave us an extensive knowledge about the microbiota that lives in the machinery of the three wineries. We highlighted the presence, in two of the valued wineries, of notable amounts of yeast inside the pipes and pumps, and in the third winery a high level of fermentative yeasts was detected in the fermentation tanks. In the case of the wineries whose pipes harbored a certain diversity of yeasts, the presence of Wickerhamomyces anomalus and Meyerozyma guilliermondii were detected as the dominant species. Their activity is common in the initial and intermediate stages of fermentation. Their contribution is known to enhance the sensory characteristics of wines, through the release of esters and other aromatic varietal compounds. However, the community of bacteria in these samples was dominated by species of undesirable acetic bacteria such as Gluconobacter, Acetobacter, Gluconoacetobacter and Paenibacillus. The yeast species found in the fermentation tanks were mainly Saccharomyces and Metschnikowia. of the resident microbiota in the winery identified the points where specific cleaning and disinfecting protocols had to be implemented to improve the hygienic conditions and guarantee the control over the fermentative processes. 21

23 Would you leave fermentation to a machine alone? A high production winery and internationally recognized brand that grows two hundred hectares of vineyards in Castilla La Mancha (Spain). Recurring fermentations stops were detected in several of a winery s 100,000 liter tanks that had acquired a generation biomass reactor as an inoculum from commercial yeasts. The winery attempted to reduce the winemaking costs, autonomously producing a commercial yeast for use in fermentation, in order to obtain homogeneous and better defined wines. Microbiology of the case Fermentative species: Saccharomyces cerevisiae Saccharomyces cerevisiae Non-Saccharomyces yeasts Other yeasts A series of WineSeq tests were conducted to evaluate the microbial populations that were actually being used as inoculum after passing through the reactor, as well as their evolution during fermentation. The tests concluded that a high concentration of acetic bacteria in the starting inoculum and the Saccharomyces cerevisiae population was much lower than required to guarantee its implantation in the process. Likewise, an implantation study was conducted on the fermented liquid, finding that the levels of implantation of the inoculated strain were low (in all cases less than 50%). In light of the systematic observation of this problem in the winery, WineSeq recommended returning to conventional inoculation practices, starting from commercial Active Dry Yeast, or conducting spontaneous fermentations with strict control of the grapes microbiological quality. 22

24 Solutions for abnormal fermentation in the presence of Brett A small production and internationally recognized winery that produces single wines with a high level of differentiation in Napa Valley. A slowing of the final phase of the fermentation kinetics was detected in a 5,000 liter tank of Merlot. The free sulfur level was low (less than 5 mg / L), and the ph higher than desired. The winemaker detected the appearance of volatile phenols, whose aromatic descriptors evoke barnyard animals. Microbiology of the case Problematic yeast: Brettanomyces bruxellensis YEAST CONCENTRATION 5, Cels/mL Saccharomyces yeasts 34,1% Brettanomyces bruxellensis Non-Saccharomyces yeasts A series of WineSeq tests were conducted to evaluate the microbial populations in the fermentation liquid and identify the reasons for the slowdown of the process. The presence of a growing and active Brettanomyces bruxellensis population was detected, which seemed to be displacing, or at least inhibiting the development, of the strains of Saccharomyces cerevisiae. This meant the stoppage of fermentation and the risk of sensory deviation. As a result of the WineSeq analysis, an increase in the SO2 dose was recommended to stop the development of Brettanomyces, as well as making a pied de cuve from another tank to accelerated the beggining of fermentation. 23

25 Lactic bacteria sometimes plays tricks A small production and internationally recognized winery that produces organic wines and embraces a sustainable viticulture. There was a stop in the final phase of the alcoholic fermentation (around 9 g / L of residual sugar) in a 10,000 liter tank of Tempranillo. An increase in the lactic acid production began, as well as in volatile acidity. Given the suspicion of an anticipation of the malolactic fermentation, which would complicate the process developed by Saccharomyces, the WineSeq team decided to study the distribution of the bacteria and yeast populations present in the fermentation liquid. Microbiology of the case Problematic bacteria: Lactobacilus brevis Saccharomyces yeasts YEAST CONCENTRATION 7, Cels/mL 7,2% Lactobacillus brevis Non-Saccharomyces yeasts A series of WineSeq tests were conducted and the presence of heterofermentative bacteria of lactic acid were detected: Lactobacillus brevis and Lactobacillus buchneri. Therefore, a start of lactic wine spoilage was diagnosed and the application of lysozymes in adequate doses for the elimination of lactic acid bacteria was recommended, decreasing the risks of volatile acidity and total acidity increase. By performing WineSeq tests during fermentation, corrective measures were able to salvage the wine. 24

26 Biogenic amines strike again; high ph is their greatest partner A high production and internationally recognized winery that makes organic wines from Pinot noir in Sonoma (California). We conducted a study of the production of biogenic amines in red wine elaboration, with commonly elevated phs, during the malolactic fermentation and during the subsequent aging of Pinot noir wines. The origin of the amines was due to the metabolism of certain bacteria and can be negative for the health and well-being of the consumer in high concentrations, due to their toxicological power. As a result of this study, a significantly higher concentration of histamine and putrescine was observed in one of the tanks. Saccharomyces yeasts Microbiology of the case Problematic bacteria: Pediococcus YEAST CONCENTRATION 5, Cels/mL 23,4% Pediococcus Non-Saccharomyces yeasts A series of WineSeq tests were conducted to evaluate microbial populations in the malolactic fermentation processes and the residual populations during aging. The dominance of lactic bacteria Pediococcus was detected in the tank, which could be responsible for the generation of the abnormal levels of biogenic amines. Some measures were proposed to reduce the contents of biogenic amines: - Maintain very good hygienic conditions in the cellar to exclude undesirable microorganisms. - Do not add extra nitrogen if there is already enough. - Manage the aging of wines taking into account the population of bacteria present in them. - Inoculate selected commercial bacteria to prevent the accumulation of amines. 25

27 Don t blame everything on Brett A medium production winery that makes high range wines from Tempranillo in Ribera del Duero. The winery detected an alteration in taste of an aged wine. There was a slight aroma associated with volatile phenols. The aromatic descriptors were identified as a barnyard animal aroma, commonly associated with Brettanomyces. Microbiology of the case Problematic yeast: Pichia manshurica Saccharomyces yeasts YEAST CONCENTRATION 3, Cels/mL 12,7% Pichia manshurica Non-Saccharomyces yeasts After performing several classical microbiological analyses for the detection of Brettanomyces, with recurring negative results, the winery conducted a WineSeq test to analyze the microbial populations in the wine. In the sample analyzed, in fact, the presence of Brettanomyces bruxellensis was not detected, which showed that the organoleptic variation of the wine was not caused by the microorganism. However, the WineSeq analysis detected, unlike conventional analytics, the presence of the yeast Pichia manshurica, an unconventional yeast, which has already been described as a contaminant in certain organic wines. The yeast was able to liberate volatile phenols such as 4-ethylphenol and mainly 4-ethylguaiacol, causing deviations similar to those produced by Brettanomyces. Furthermore, the detected yeast was able to produce a superficial veil on the wine, as well as a biofilm on the surfaces of the barrels and the fermentation or storage tanks, acting as a potential source of contamination in the cellar. Although this yeast is, in general, sensitive to potassium metabisulfite and detergent products, the WineSeq team recommended extracting the wine and discarding the barrel as a preventive measure. 26

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29 3 DIFFERENTIATION CASES Being able to justify differentiating your wine from the rest may be the key to success in the market. Many wineries have used WineSeq technology to identify the origin of the differentiation of their product. The origin is always the microbial community that lives in the vineyard. CASE 1 Are you ready to read your microbial footprint? CASE 2 Microbial biodiversity as a new terroir differentiator CASE 3 Tell me the microbiota of your cellar and I will tell you who you are 28

30 Are you ready to read your microbial footprint? A selection of twenty-nine wineries worldwide were chosen to develop a differentiation study of the microbiology of soils. We conducted a WineSeq test of three hundred and eighteen soils of twenty-nine wineries from fourteen wine regions worldwide. The main objective was the identification of species that characterize the microbial footprint in different production areas. This study is based on the use of WineSeq for the analysis and evaluation of the results obtained, as well as the definition of this technology as a terroir biomarker. Microbiological patterns were observed to group soils according to their geographical origin and management. This study demonstrated the value of WineSeq testing for the definition of terroir and the influence of vine growing practices. The microbiome is a measurable and quantifiable parameter of the microbiological species that promotes differentiation for its biodiversity. Likewise, it becomes an instrument to delimit certain areas of production regarding the qualitative parameters that will influence the elaborations of the plots. It also becomes a tool to create value in the Appellation of Origin and create a better link with the terroir. 29

31 Microbial biodiversity as a new terroir differentiator A small production and internationally recognized winery that makes single wines in Rioja Alavesa. The winery requested the microbial biodiversity characterization of each plot through WineSeq tests to comply with the new Appellation regulation and the zoning project of their vineyards. The results are of interest for the winemaking of single wines, adapting viticultural and oenological practices to each plot to maximize their potential. Microbiological patterns associated with the diversity of soils and management that characterized the different plots were identified. Though indistinguishable to the naked eye, one of the plots showed a unique and differentiated biodiversity pattern from other plots, despite similar edaphic factors. WineSeq testing provided a hightened level of knowledge of the characteristics of this plot and increased the ability to compare it to other nearby plots. Thereby, the WineSeq test was able to confirm its authenticity and strengthen the case for the request for certification of Single Vineyard. This demonstrates the usefulness of WineSeq technology for the characterization of soils from a microbiological point of view as a measurable and quantifiable tool. The microbiome has been established as a basic unit for the certification of a geographical area based on microbiological species that promote differentiation. 30

32 Tell me the microbiota of your cellar and I will tell you who you are A small production and internationally recognized winery that makes organic wines in Napa Valley. seeks to the characterize the resident microbiota in the cellar (surfaces, ducts and machinery) in order to evaluate whether the sanitation methods that were carried out were sufficient to avoid the formation of reservoirs of bacteria and yeasts that could act as contaminants in future vintages. A WineSeq test protocol was established in the winery after the end of the harvest and the conditioning and cleaning of the facilities, carrying out sampling at the following points: the hopper, the press, the pipes, the pumps and the fermentation tanks. A WineSeq study was conducted to determine the microbiota of the winery based on the established protocol. The set of surfaces and machinery showed reasonable levels of asepsis or hygiene for a winery, however, it highlighted a community of acetic bacteria (Gluconobacter and Acetobacter) and certain yeasts (Metschnikowia and Zygosaccharomyces) in the pipes and one of the pumps, as well as in one of the tanks analyzed. The resident microbiota study in the winery identified the points where specific cleaning protocols should be implemented to improve hygienic conditions and ensure control over fermentation processes. 31

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34 4 BIOREMEDIATION CASES Are you going to apply a treatment or a product to your vineyard and you are not sure about how it can affect it? Have you already applied it and you have noticed imbalances in the vine or even in the processes in the winery? Discover how some winegrowers have solved their problems knowing the microbiology of the products that they apply. CASE 1 Don t be shy and choose the best fertilizer for your plot CASE 2 Feel free to apply phytosanitary treatments, but be careful! 33

35 Don t be shy and choose the best fertilizer for your plot A small production and high-value winery that makes wines from a single plot. We developed a protocol to study the application of an organic fertilizer in several vineyards in reference to a sign plot. The trial was carried out in triplicate and in plots with different terroirs, in order to assess the evolution of microbial species in soils. The use of organic fertilizers to improve the nutritional properties of soils is a current trend that indeed contributes to maintaining diversity and sustainability. WineSeq concluded that organic corrections applied to soils tend to reduce the population of the microbial species present in them. This decrease also occurs in the pathogens that affect the vines. On the other hand, the increase of yeasts and bacteria with oenological interest was detected, as well as nitrogen fixing species, although the appearance of species or biocontrol agents was not observed. 34

36 Feel free to apply phytosanitary treatments, but be careful! A vine-growers association wanted to test its members vineyards to improve sustainable viticulture. WineSeq developed a protocol for the evaluation of fifty-two vineyard soils. The interpretation of our results is based on the criteria established by the vine-growers Association. Each case was studied individually and the necessary information was gathered to conduct a WineSeq test for defining the diversity of microbial populations. According to the initial documentation phase, we identified a plot with an aggressive phytosanitary application program with chemical products. The analysis of the vineyard soil indicated a very reduced microbial diversity, as well as the presence of a fungal pathogen associated with Black Foot disease. A resistance to the phytosanitary treatments that had developed and caused problems in the two previous years, and was starting to extend to bordering plots. Likewise, no presence of bacteria or yeast species of oenological interest was detected. Once analyzed all the factors and the results obtained from the WineSeq test, our conclusion was that this soil was at risk of suffering a microbial sterility and the consequent deterioration of the productive life and the sustainability of the crop, so so it is recommended to apply bioremedation techniques. 35

37 WINESEQ WORKS! This collection of success cases is a proof that guarantees the operability of WineSeq and its practical applications. However, it is not the only one. Biome Makers has been awarded and recognized many times by some of the most prestigious institutions in the field of innovation and AgTech. WINnovation Award 2017 Semi-finalist InnoSTARS Competition AgFunder Innovation Awards 2016 Finalist Agtech Latam Chile 2017 Pyme Innovadora 2017 Finalist EU ehealth Competition 36

38 C R E A T O R S Biome Makers, USA 665 3rd St #250 San Francisco, CA USA info@biomemakers.com +1 (415) Biome Makers, EUROPE Paseo de Belén Nº Valladolid Spain info@biomemakers.com (+34)