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Bacteria of vineyard and terroir, and presence of Oenococcus in Priorat (South Catalonia) grapes

2nd May 2015 

The vine growers believe that the land on which they grow vines gives the wines a unique quality, and that is called terroir. We can consider that the physiological response of the vines to the type of soil and climatic conditions, together with the characteristics of the variety and form of cultivation, result in a wine organoleptic properties that define their terroir (Zarraonaindia et al 2015 ). However, it is not known if there could be a very specific microbiota of each terroir, as this subject has been barely studied.

Wine microorganisms in the grapes? Saccharomyces is not there or it has not been found there

The main protagonists of wine fermentations, alcoholic one (yeast Saccharomyces cerevisiae) and malolactic one (lactic acid bacteria Oenococcus oeni) usually do not appear until the must grape is fermenting to wine, in the cellar. In normal healthy grapes, S. cerevisiae is hardly found.

Oenococcus oeni in the grapes ? We have found it !

Regarding O. oeni, so far very little has been published about its presence and isolation from the grapes. In some works, as Sieiro et al (1990), or more recently Bae et al (2006), some lactic acid bacteria (LAB) have been isolated from the surface of grapes, but not O. oeni. Only Garijo et al (2011) were able to isolate a colony (only one) of O. oeni from Rioja grapes. Moreover, DNA of O. oeni has been detected in a sample of grapes from Bordeaux (Renouf et al 2005, Renouf et al 2007) by PCR-DGGE of rpoB gene, although in these works no Oenococcus has been isolated.

I am pleased to mention that recently our team have managed to isolate O. oeni from grapes, and typify them, and we are now working on a publication about it (Franquès et al 2015). Indeed, our research team of lactic acid bacteria (BL-URV), together with colleagues working on yeasts from the same group “Oenological Biotechnology” (Faculty of Oenology at the Universitat Rovira i Virgili in Tarragona, Catalonia, Spain) is working on a European project, called “Wildwine “(FP7-SME-2012 -315065), which aims to analyse the autochthonous microorganisms of Priorat area (South Catalonia), and select strains with oenological potential. This project also involves the Priorat Appellation Council and the cellar Ferrer-Bobet, as well as research groups and associations wineries from Bordeaux, Piedmont and Greece. In the framework of this project we took samples of grapes (Grenache and Carignan) from several vineyards of Priorat (Figure 1), as well as samples of wines doing malolactic fermentation. From all them we got 1900 isolates of LAB. We optimized isolation from grapes from the pulp and juice with various methods of enrichment, and so we got 110 isolated bacteria from grapes, identified as O. oeni by specific molecular techniques. Once typified, we have found that the molecular profiles of these strains do not coincide with commercial strains and so they are autochthonous. In addition, some of these strains from grapes were also found in the corresponding wine cellars.

Fig 1 garna-cari Priorat

Figure 1. Taking samples of Grenache (left) and Carignan (right) in Priorat area to isolate lactic acid bacteria such as Oenococcus (Pictures Albert Bordons).

The microbiota of grapes

The grapes have a complex microbial ecology, including yeasts, mycelial fungi and bacteria. Some are found only in grapes, such as parasitic fungi and environmental bacteria, and others have the ability to survive and grow in wines: especially yeasts, lactic acid bacteria (LAB) and acetic acid bacteria. The proportion of all them depends on the maturation of the grapes and the availability of nutrients.

When the fruits are intact, the predominant microbiota are basidiomycetous yeasts as Cryptococcus and Rhodotorula, but when they are more mature, they begin to have micro fissures that facilitate the availability of nutrients and explain the predominance just before the harvest of slightly fermentative ascomycetes as Candida, Hanseniaspora, Metschnikowia and Pichia. When the skin is already damaged more damaging yeasts may appear, as Zygosaccharomyces and Torulaspora, and acetic acid bacteria. Among the filamentous fungi occasionally there may have some very harmful as Botrytis (bunch rot) or Aspergillus producing ochratoxin. Although they are active only in the vineyard, their products can affect wine quality.

On the other hand, environmentally ubiquitous bacteria have been isolated from the grapes skin, as various Enterobacteriaceae, Bacillus and Staphylococcus, but none of them can grow in wine (Barata et al 2012).

Coming back to the possible specific microbiota of terroir, it has been found that some volatile compounds contributing to the aroma of the wine, such as 2-methyl butanoic acid and 3-methyl butanol, are produced by microorganisms isolated in the vineyards, as Gram-positive bacterium Paenibacillus, or the basidiomycetous fungus Sporobolomyces or the ascomycetous Aureobasidium. Therefore, there could be a relationship between some of the microbial species found in grapes and some detected aromas in wine, coming from the must of course (Verginer et al 2010).

Metagenomics as analytical tool of microbiota from grapes

Since conventional methods of isolation and cultivation of microorganisms are slow, laborious and some microbes cannot be grown up in the usual isolation media, massive sequencing methods or metagenomics are currently used. These consist of analysing all the DNA of a sample, and deducing which are the present microorganisms by comparing the sequences found with those of the databases. For bacteria the amplified DNA of V4 fragment from 16S RNA gene is used (Caporaso et al 2012).

This technique has been used with samples of botrytized wines (Bokulich et al 2012) and various LAB have been found (but not Oenococcus), including some not normally associated with wine. It has also been used to see the resident microbiota in wineries and how it changes with the seasons, resulting that in the surfaces of tanks and machinery of the cellar there is a majority of microorganisms neither related with wine nor harmful (Bokulich et al 2013).

With this technique Bokulich et al (2014) have also analysed the grapes and they have seen clear differences between the proportions of bacterial groups (and fungi) from different places, different varieties, as well as environmental or bio geographical conditions. For example, when analysing 273 samples of grape musts from California, the 3 varieties (Cabernet, Chardonnay and Zinfandel) are quite discriminated in a principal components analysis with respect to the bacterial communities found in each sample (Figure 2).

Thus, the dominant bacterial taxa or groups in a variety or given environment could provide some specifics traits on those wines, and this could explain some regional or terroir patterns in the organoleptic properties of these wines (Bokulich et al 2014).

Fig 2 ACP Bokulich 2014

Figure 2. Principal component analysis of bacterial communities of grape musts samples of Sonoma (California) from 3 varieties (Cabernet in red, Chardonnay in green and Zinfandel in blue) (Bokulich et al 2014).

We have also carried out a massive sequencing study with the same grape samples from which we have obtained isolates of O. oeni, as said before (Franquès et al 2015), and in more than 600,000 analysed sequences of 16S rRNA, we have found mainly Proteobacteria and Firmicutes. Among these gram-positive, we have found sequences of lactic acid bacteria (15%) and from these we have successfully confirmed the presence of O. oeni in 5% of the sequences. Therefore, we have isolated O. oeni from grapes and we have detected their DNA in the samples.

The bacterial microbiota of the vineyards and soil

As we see, microbiota of grapes and wine has been studied a little, but the soil microbiota has not been characterized. This one can define more clearly the terroir, which is influenced by the local climate and characteristics of the vineyard.

In Figure 3 the main genera found in different parts of the vine and soil are summarized (Gilbert et al 2014).

Fig 3 Gilbert 2014

Figure 3. Main bacteria and fungi associated with organs and soil of Vitis vinifera (Gilbert et al 2014)

Recently an interesting scientific work (Zarraonaindia et al 2015) has been published on this subject, with the aim to see if the soil could be the main original source of bacteria that colonize the grapes. These authors took samples of soil, roots, leaves, flowers and grapes from Merlot vines, from different areas and years, of Suffolk, New York, and they analysed the bacterial DNA by 16S rRNA sequencing. They found that 40% of the species found were present in all samples of soil and roots, while there was more variability in leaves and fruits, and moreover, 40% of those found in leaves and fruits were also found in soils. All this suggests that many bacteria originate in the soil.

Regarding the type of bacteria, they found that Proteobacteria (especially Pseudomonas and Methylobacterium) predominated (Figure 4), mainly in the aerial parts of the plant. There were also Firmicutes as expected, and Acidobacteria and Bacteroides.

Fig 4 microbiota vineyard

Figure 4. Composition of the bacterial community, at Phylum level, in samples from different organs of the vine and its soil (Zarraonaindia et al 2015).

Although variations were observed in all samples depending on the year (there may be different climatic conditions) and according to different edaphic factors (pH, C: N, humidity), the principal-components analysis (Figure 5) showed that the main types of samples (soil, roots, leaves, grapes) differ quite well, and bacterial taxon composition in samples of grape juice before fermentation is similar to that of grapes.

Fig 5 distribució grups mostres OTUs

Figure 5. Principal-components analysis showing the similarities in terms of the composition of bacterial taxonomic groups, among sample types, including musts (Zarraonaindia et al 2015).

This suggests that the bacterial community found in grapes remains relatively stable until the processing to musts, and that it is more stable than the differences between organs. At the same time, a large number of representatives of bacterial phyla of the grapes come from the soil. This can be explained because when grapes are harvested by hand, they are often placed in boxes that are left on the ground, or for mechanical harvest, the machinery used removes the soil and generates dust, which can colonize the grapes.

Therefore, the soil microbiota is a source of bacteria associated with vines and may play a role in the must and therefore in the wine, and potentially in the formation of the terroir characteristics. Some of these bacteria may have some roles not yet known in productivity or disease resistance of the plant, or contribute to the organoleptic characteristics of wine (Zarraonaindia et al 2015).

In addition, and thinking in wine microorganisms responsible for fermentations, as said, in our laboratory we have confirmed that there are some O. oeni strains in grapes and we have confirmed this by detecting their DNA in the same grapes.


Bae S, Fleet GH, Heard GM (2006) Lactic acid bacteria associated with wine grapes from several Australian vineyards. J Appl Microbiol 100, 712-727

Barata A, Malfeito-Ferreira M, Loureiro V (2012) The microbial ecology of wine grapes (Review). Int J Food Microbiol 153, 243-259

Bokulich NA, Joseph CML, Allen G, Benson AK, Mills DA (2012) Next-generation sequencing reveals significant bacterial diversity of botrytized wine. Plos One 7, e36357

Bokulich NA, Ohta M, Richardson PM, Mills DA (2013) Monitoring seasonal changes in winery-resident microbiota. Plos One 8, e66437

Bokulich NA, Thorngate JH, Richardson PM, Mills DA (2014) Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. PNAS nov 25, E139-E148

Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621–1624

Franquès J, Araque I, Portillo C, Reguant C, Bordons A (2015) Presence of autochthonous Oenococcus oeni in grapes and wines of Priorat in South Catalonia. Article in elaboration.

Garijo P, López R, Santamaría P, Ocón E, Olarte C, Sanz S, Gutiérrez AR (2011) Eur Food Res Technol 233, 359-365

Gilbert JA, van der Lelie D, Zarraonaindia I (2014) Microbial terroir for wine grapes. PNAS 111, 5-6

Renouf V, Claisse O, Lonvaud-Funel A (2005) Understanding the microbial ecosystem on the grape berry surface through numeration and identification of yeast and bacteria. Aust J Grape Wine Res 11, 316-327

Renouf V, Claisse O, Lonvaud-Funel A (2007) Inventory and monitoring of wine microbial consortia. Appl Microbiol Biotechnol 75, 149-164

Sieiro C, Cansado J, Agrelo D, Velázquez JB, Villa TG (1990) Isolation and enological characterization of malolactic bacteria from the vineyards of North-western Spain. Appl Environ Microbiol 56, 2936-2938

Verginer M, Leitner E, Berg G (2010) Production pf volatile metabolites by grape-associated microorganisms. J Agric Food Chem 58, 8344-8350

Zarraonaindia I, Owens SM, Weisenhorn P, West K, Hampton-Marcell J, Lax S, Bokulich NA, Mills DA, Martin G, Taghavi S, Van der Lelie D, Gilbert JA (2015) The soil microbiome influences grapevine-associated microbiota. mBio 6, e02527-14


1st Conference on Research in Viticulture and Oenology in Catalonia

First of all, I apologize to friends of this blog for these last five months without any post. Sorry, I had too much work of teaching and research, and also I had to prepare this Conference that I am commenting now.

I have had the pleasure of being the organizer of this conference, along with Pep Llauradó, because I am the coordinator (and Pep is the manager) of the sub-campus Oenology of CEICS, the Campus of International Excellence Southern Catalonia.

CEICS is the strategic aggregation, driven by the Universitat Rovira i Virgili, of various institutions and structures of teaching, research, transference, and the productive sector of southern Catalonia, with the goal of becoming an international benchmark in 5 areas: Chemistry, Nutrition-Health, Tourism, Culture-Heritage and Oenology.

In 2012 I had the honor of being named coordinator of subcampus Oenology of CEICS, by the rector of the URV Xavier Grau, who is the president of CEICS.

logo CEICS Eno

The subcampus Oenology includes URV, Fundació URV, the technological park VITEC of Falset, IRTA, INCAVI, the cluster INNOVI, and most of catalan DO (Appelation of Origin), especially those of Tarragona, and companies and wineries, such as Freixenet and others.

One of the main objectives of CEICS is to promote the visibility of research done in the region. Therefore, since the beginning of CEICS, with the occasion of the 1st Forum held in November 2011, a conference-meeting like this was already planned, in order to share and diffuse the research done in Catalonia.

foto Jornada

Inauguration of the Conference, with the rector of the URV Xavier Grau and the Dean of the Faculty of Oenology Joan Miquel Canals.

In Catalonia we have often the opportunity to attend various scientific conferences or technical meetings on oenology and/or viticulture. The INCAVI, the Catalan Association of Winemakers (ACE) or the cluster INNOVI, or the Facultat d’Enologia from URV often organize special courses or seminars. All of them are very interesting and necessary, and add value to the Catalan wine industry, but perhaps there had not yet been done so far a meeting of all catalan researchers in this field, in order to see all the research that is being carried out, not just in a specific focus or subject.

retol Jornada 3

All in all, we considered appropriate to take the energy and push of CEICS for organizing this conference. The main objective was to publicize and disseminate the research carried out in Catalonia in Oenology and Viticulture and related issues. Before the conference, we reviewed databases of scientific publications in the last years, and we have seen that in Catalonia there are about 20 research groups whose main lines are related to the sciences of oenology and viticulture, but in addition there are 40 other groups and researchers that although his main area of research is not the viticulture and oenology, in recent years they have published several papers related to aspects of vitiviniculture, even from scientific disciplines apparently far away. All they were invited to this conference held in Tarragona (at Campus Sescelades URV), to present their most innovative research in the form of posters. I must thank them all for the good response, since there was a total of 44 posters presented.




Another implicit goal of the conference was meeting people. This conference was an opportunity to meet, discuss and explore the details of recent work with colleagues from different centers in Catalonia, maybe even meeting in person for the first time in some cases, and promote collaboration between groups.



But in addition, this Conference has not been a meeting exclusively for scientists. The research is meaningless if it has no practical application and benefits in the winemaking process, though often not immediate implementation. It binds with the main objective of the conference, as mentioned, to make visible the research done here. In this regard, some of the posters that were presented have been selected so that the authors briefly commented on the general sessions, and the selection has been done keeping in mind the interest of the research done for the wine industry.

Most of the program of the day was related to the same posters presented. So in addition to seeing and commenting particularly with authors throughout the day, there were four general sessions where experts presented them in summary. In these four sessions grouping of posters was based on their areas.



Robert Savé and Anna Puig


Lluís Tolosa taking notes of one poster.

Robert Savé (researcher of IRTA) presented and moderate the session of Viticulture. Olga Busto (professor of URV) did the same with the Oenological Chemistry and Technology session. Anna Puig (researcher of INCAVI) did the same in session of Microbiology in Enology and Viticulture, and finally the sociologist and writer Lluís Tolosa presented and moderate the session of other subjects related to the vine and wine, such as the issues of effects on health, environmental impact, socioeconomic aspects like tourism, food industry or other applications, or historical and cultural aspects and even prehistoric.

The collection of papers presented showed good international quality of research conducted in oenology and viticulture in Catalonia, and at the same time, the wide variety of topics. With the abstracts of papers a book has been edited: “1st Research Conference on Enology and Viticulture in Catalonia, Book of abstracts of the conference organized by CEICS, Tarragona June 4, 2013”, which has been published by Service of the Publications URV, with ISBN 978-84-695-7878-0.

In addition, the conference included the inaugural lesson of the internationally renowned researcher in vine genetics, José Miguel Martínez Zapater, who has an extensive curriculum research in plant genetics.

foto JM Mz Zapater

Since 1998 Dr. Zapater works on the genetics of Vitis, having achieved important results, both on varietal identification by molecular techniques and also on vine transcriptomics, with the study of gene expression in function on the conditions of climate change. Since 2010 is the director of the new Instituto de Ciencias de la Vid y el Vino (ICVV) in Logroño.


The lesson of Dr. Zapater was an excellent review of the genetic variability of Vitis and of the molecular techniques to identify different vine varieties.

We can say that the Conference, with about 70 attendees, was a success. There were researchers, students, technicians and professionals, of URV, INCAVI, IRTA, VITEC, UAB, UB, UPC, UDL, CSIC, and seven companies. In a survey that we asked them to fill up, we have concluded that most of the participants were very satisfied with the conference, its organization, its development, and express their wishdom to do it again, annualy or every two years.

jornada + logo ceics

Climate change and wine: the Spanish project CENIT DEMETER has ended


Please go the original post in catalan: “Canvi climàtic i vi: finalització del projecte CENIT DEMÉTER”

and use the translator buttons with flags, at right corner.

Thanks for your collaboration

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