Yeast biodiversity evolution over decades in Dealu Mare-Valea Calugareasca vineyard

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1 Romanian Biotechnological Letters Copyright 2011 University of Buchare Vol. 16, No.1, 2011, Supplement Printed in Romania. All rights reserved ORIGINAL PAPER Yeast biodiversity evolution over decades in Dealu Mare-Valea Calugareasca vineyard Abstract Received for publication, November 6, 2010 Accepted, February 11, 2011 MATEI RADOI FLORENTINA 1, BRINDUSE ELENA 2, NICOALE GETUTA 1, TUDORACHE AURELIA 2, TEODORESCU RAZVAN 3 1 University of Agronomical Sciences and Veterinary Medicine of Bucharest, Faculty of Biotechnology 59, Marasti Ave., Bucharest, Romania, florentina.matei@biotehnologii.usamv.ro 2 ICDVV Valea Calugareasca, Prahova, Romania 3 University of Agronomical Sciences and Veterinary Medicine of Bucharest, FIFIM Belonging to a wider national project of yeast biodiversity conservation from consecrated Romanian vineyards, this study has been performed in a red wine recognized area, Valea Calugareasca from Dealu Mare County. The main goal has been to isolate yeasts from the vineyard, from the grape and during different phases of alcoholic fermentation and to perform their identification. The yeast isolation has been done from vine plantation of Cabernet Sauvignon, Merlot, Feteasca Neagra and Pinot Noir grape wines. We have used classical methodology for isolation and purification on YEPD, finally obtaining 262 isolates. The identification has followed two approaches: one by classical morphological and cultural analysis, using Barnett J.L. (2000) guide and the second one, employed rapid physiological tests of API 20 C Biomerieux galleries. A comparison between the two identification methods has been performed. A secondary goal of our study, but very important in the context to microbiota evolution, has been to compare our results ( ) to former results obtained by Dr. Kontek A. in the years in the same areal. Because the former work was performed by classical identification methods, for a realistic comparison we have used the same methods. We should have taken into account also the changes in the wine yeast taxonomy. During the last 4 decades, the yeast biodiversity have changed, by the species number increase. In the years 70 s in the beginning of the fermentation non-saccharomyces species were Kloeckera apiculata (40%) and Pichia norvegensis (14%), while in the last 3 years new species have been identified, as Candida famata (3), Debaryomyces hansenii (2). During the middle of the fermentation, Saccharomyces species were dominant, as it was expected. Keywords: wine yeast, biodiversity evolution, Dealu Mare vineyard Introduction In the field of wine yeast, main studies have been usually focused on Saccharomyces strains as being responsible for the alcoholic fermentation. But, former or more recent works, proves the involvement in this process of other non-saccharomyces yeasts during the initial steps, while they have an important influence on the final organoleptic properties of the wines (Pretorius, 2000). The role of non-saccharomyces species in wine industry may be an interesting debating subject for the researchers. Initially, it has been thought that this species die during the first phase of the alcoholic fermentation because of the high alcoholic content due the Saccharomyces cerevisiae activity. The last decade s researches have proved that non- Saccharomyces species can survive during the alcoholic fermentation and can reach a population of CFU/ml. These species can improve the final wine organoleptic properties, mainly on aromatic level, by producing specific enzymes and volatile products for different vineyard [Zohre, 2002). In the last years has been reported the isolation during the 113

2 MATEI RADOI FLORENTINA, BRINDUSE ELENA, NICOALE GETUTA, TUDORACHE AURELIA, TEODORESCU RAZVAN alcoholic fermentation of non-saccharomyces species (Hanseniaspora, Candida, Kluyveromyces) of high ethanol tolerance, similar to Saccharomyces cerevisiae [Pina et al., 2004; Xufre et al., 2006; Nisiotou et al., 2007]. Belonging to a wider national project of yeast biodiversity conservation from consecrated Romanian vineyards, this study has been performed in a red wine recognized area, Valea Calugareasca from Dealu Mare County. The main goal has been to isolate yeasts from the vineyard, from the grape and during different phases of alcoholic fermentation and to perform their identification. Wine production in most countries is based on the use of commercial strains leading to the colonization of the wineries by these strains with the consequent reduction of autochthonous biodiversity. The vineyard could be an important source of native yeasts of oenological interest. The specific yeast microbiota biodiversity study in a DOC winemaking areal as Dealu Mare Valea Calugareasca is an important goal for the fundamental research and moreover to define the wine characteristics ( terroir aspects) in the area. In Romania, few works had reported the biodiversity of yeast in different vineyards during the years 50 s and 60 s (Lepadatu V, Septilici G, Teodorescu S), but a large study has been done in Dealu Mare region by Dr. Kontek in 70 s. Only in the 90 s Dr Burcea M. has started a new era in the wine yeast study in Romania, isolating valuable oenological strains from Panciu Region [Burcea, 1998; Burcea, 2005]. In this regard, another goal of our study, but very important in the context of microbiota evolution, has been to compare our results ( ) to former results obtained by Dr. Kontek A. in the years in the same areal. Because the former work was performed by classical identification methods, for a realistic comparison we have used the same methods. Materials and methods The origin of the yeasts has been Dealu Mare (Grand Hill in Romanian language) vineyard, Valea Calugareasca center (Fig.1). Dealu Mare vineyards make up the most compact Romanian vine culture and wine making area (spreads on approximately 400 square kilometers over sub-carpathian Hills). Dealu Mare is the homeland of red wines, where the red grapes found those suitable conditions that would stimulate their qualities. A number of factors are responsible for this: the plantations stretch over hill-slopes facing the light and heat of the sun and looking south and south-east, the temperature is higher during the period of vegetation of the vines, the rain does not fall in excess, the soils are not very rich and contain a high level of iron oxides. Figure 1. Dealu Mare wine area in the Romanian vineyards assembly (squared area) As biologic material have been used grape varieties for red wines recommended in Valea Calugareasca centre, respectively Cabernet Sauvignon (ecological and non-ecological plantation), Merlot, Feteasca Neagra and Pinot Noir. Sampling and yeast isolation. The sampling has been performed during 3 harvest years, respectively 2007, 2008 and The grape berries have been harvested in September 114 Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011)

3 Yeast biodiversity evolution over decades in Dealu Mare-Valea Calugareasca vineyard and October with pedicel. For each grape variety have been harvested samples from 3 points of the plot, from different levels of the rope: ground, middle and top. The samples have been prepared in two ways. For the first series the berries have been harvested in aseptic conditions and introduced in an Erlenmeyer with sterile distillated water in order to collect the yeast from the grape surfaces. After 24 hours of shaking, from the suspension, plates with YEPD have been inoculated and cultivated during hours at 27 o C. For the second series, the berries have been crushed in aseptic conditions and the natural fermentation has occurred. During the fermentation, three moments for sampling have been chosen: beginning, middle and final phase of the fermentation. After applying Domerq dilutions, the samples have been inoculated on YEPD. Every isolated colony has been passed in slant on GYP medium and pure cultures have been obtained which were the identification subjects. Yeast identification and characterization. Two approaches have been followed for the identification: classical morphological and cultural tests and rapid biochemical tests. In the case of classical identification there have been examined morphological and cultural aspects (shape and cell dimension of the cell) of each strain on liquid media after 3 days cultivation on Wickerham media at 25 o C and on solid media YEPD; also, the pseudomycelia formation on potato-agar media after 12 cultivation days have been observed; another parameter was the sporulation on Gorodkova media after 30 cultivation days. The physiological characters have been studied by rapid test API 20 C AUX (Biomerieux), which allows testing the assimilation of 19 substrates (Fig.2). The obtained data have been analyzed by the APILAB PLUS software. Figure 2. Aspects of API 20 C galleries (yeast physiological identification) In recent years, to improve the conventional methods, rapid kits for yeast identification have been developed. However, they were initially designed for clinical diagnosis and their application is restricted to yeast species of medical interest [Deak,1993]. The reproducibility of these techniques is questionable due to the fact that they depend on the physiological state of the yeast cells. When the API 20C tests have not been clear, classical fermentation and assimilation tests have been done for sugars and nitrates. The taxonomical identification has been performed following the Barnett J.A. (2000) yeast characterization. Results and discussions Yeast Biodiversity in Valea Calugareasca Center- harvests Yeasts are unicellular fungi and can be classified into two phylogenetic groups, teleomorphic and anamorphic ascomycetous yeasts and teleomorphic and anamorphic basidiomycetous yeasts [Kurtzman &Fell, 1998]. The differentiation of taxa is usually achieved by comparison of morphological traits and physiological features [Barnett et al., 2000], which in some cases can lead to an incorrect classification of species or a false identification of strains. During harvests we have isolated from Dealu Mare vineyard, Valea Calugareasca centre, more than 2000 different colonies which belong to 262 yeast strains. Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011) 115

4 MATEI RADOI FLORENTINA, BRINDUSE ELENA, NICOALE GETUTA, TUDORACHE AURELIA, TEODORESCU RAZVAN Following the classical identification procedure, from a taxonomical point of view these strains belongs to six families, 12 genus (7 genus forming spores), respectively to 24 species (Table 1). Table 1. Taxonomy of yeast strains isolated from Dealu Mare vineyard, Valea Calugareasca Centre Phylogenetic group Class Order Family Genus Species Ascomycota Hemiascomycetes Saccharomycetales Candidaceae Candida colliculosa famata lusitaniae magnoliae pelliculosa sphaerica stellata utilis Geotrichum capitatum penicillatum Kloeckera apiculata Metschnikowiaceae Clavispora lusitaniae Saccharomycetaceae Kodamaea ohmeri Pichia anomala jadinii Debaryomyces hansenii Saccharomyces cerevisiae Torulaspora delbruecki Saccharomycodaceae Hanseniaspora uvarum Basidiomycota Hymenomycetes Tremellales Tremelaceae Cryptococcus albidus neoformans Urediniomycetes Sporidiales Sporidiobolaceae Rhodotorula glutinis minuta mucilaginosa From the grape surface have been isolated 40 yeast strains. 80% belongs to Ascomycota and 20 % to Basidiomycota. The dominant species have been Kloeckera apiculata (20%), Candida famata (15%) and Debaryomyces hansenii (12.5%). It has been identified a single strain of Saccharomyces cerevisiae (2.5%) (Fig.3). Predominant species are the non-sporogene ones (85%). The species showing fermentative power represent 80 % from the identified strains. 20,0% 7,5% 1 20,0% Candida famata Candida lusitaniae Candida magnoliae Candida utilis Geotrichum capitatum Kloeckera apiculata Debaryomyces hansenii Saccharomyces cerevisiae Hanseniaspora uvarum 7,5% 10,0% 1 Cryptococcus albidus Cryptococcus neoformans Rhodotorula glutinis Rhodotorula minuta Rhodotorula mucilaginosa Figure 3. Yeast biodiversity on grape berries (harvests , Dealu Mare vineyard) In the first fermentation phase the same complex microorganism biodiversity has been noticed, but the dominant species is Candida famata (32.5%), followed by Debaryomyces hansenii (22.5%). Also, the non-sporogene species are dominant, 77.5% respectively (Fig.4) During the alcoholic fermentation the non-saccharomyces ratio decreased. The literature assumes this decrease to the yeast sensitivity to ethanol concentration, to the cell to cell contact followed to a high cells density or to an indirect toxin action (killer effect). The dominant species became Saccharomyces cerevisiae in a percentage of 50-78%. Aside these strains have been identified some medium ethanol tolerant genera such as Candida, Klavispora, Pichia, Torulaspora (Table2). 116 Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011)

5 Yeast biodiversity evolution over decades in Dealu Mare-Valea Calugareasca vineyard Rhodotorula mucilaginosa Rhodotorula minuta Rhodotorula glutinis 10,0% Saccharomyces spp. Debaryomyces hansenii Kloeckera apiculata Candida utilis Candida pelliculosa Candida stellata Candida famata 7,5% 9,8% ,05 0,1 0,15 0,2 0,25 0,3 0,35 Figure 4. Yeast biodiversity in the first alcoholic fermentation phase Our data do not contradict all recent research, which agrees that the predominant species on healthy grapes are apiculate yeasts like Hanseniaspora uvarum (and its anamorph form Kloeckera apiculata) and oxidative species such as Candida, Pichia, Kluyveromyces and Rhodotorula [Fleet, 1993; Schuller, 2005]. Contrarily, fermentative species of the genus Saccharomyces, predominantly S. cerevisiae, occur in extremely low number on healthy undamaged berries or in soils, while damaged grapes are believed to be an important source of S. cerevisiae [Mortimer, 1999]. Table 2. Yeast biodiversity during alcoholic fermentation in Valea Calugareasca Centre, Dealu Mare region (harvests ) data are expressed in % Genus Beginning of AF* Middle phase of AF End phase of AF Candida Geotrichum Kloeckera Clavispora Kodamaea Pichia Debaryomyces Saccharomyces Torulaspora Rhodotorula * AF Alcoholic Fermentation The development of non-saccharomyces species in the middle or in the ending phase of the alcoholic fermentation has been conditioned by a few factors. The first factor is the biodiversity of the initial yeast population on the grape surface, which contains 80% species with fermentative capacities (Candida sp., Debaryomyces sp., Hanseniaspora sp.). Another factor it has been the chemical composition of the grape must which may contain residues of pesticides. For example, in the ecological plantation, the dominant species was Candida utilis aside species belonging to Rhodotorula genus, respectively R.glutinis and R.mucilaginosa, while in the non-ecological plantation, the dominant was Candida famata, aside Candida lusitaniae and Rhodotorula mucilaginosa. During the first fermentation phase, in the case of the ecological plantation of Cabernet Sauvignon, there have been identified in equal proportions, Candida utilis, Kloeckera apiculata, Debaryomyces hanseni, Rhodotorula mucilaginosa and only one strain of Saccharomyces cerevisiae. In the case of non-ecological plantation, in the first fermentation phase Candidata famata kept the dominant position. Finally, another important factor are the species characteristics which can depend on the physiological and biochemical compatibility of the strain with the chemical structure of the berries skin, also may depend to the species tolerance to the environmental factors or to the species interaction [Mortimer, 1999]. Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011) 117

6 MATEI RADOI FLORENTINA, BRINDUSE ELENA, NICOALE GETUTA, TUDORACHE AURELIA, TEODORESCU RAZVAN Comparative analysis of yeast biodiversity in Dealu Mare vineyard, Valea Calugareasca Centre ( / ) From the 70 s data [Kontek, 1975] the 244 yeast isolates from Dealu Mare vineyard belong to 22 species and 7 genera, respectively, under the present taxonomy, to Endomycetaceae and Criypococcaceae; 189 strains belong to three sporogene genera and 53 to four non-sporogene genera; this classification has been done according to Lodder and Kreger van Rij, The comparison started from the data obtained by Dr. Kontek A. and his collaborators during in the same areal. Because of that time techniques (classical morphological and cultural identification) we have used in our study the same method in order to be able to compare the two data series. It has to be noticed that the conventional methodology for yeast identification requires evaluation of some tests, and the process is complex, laborious and time consuming. Also, in this regard, the biodiversity from 70 s has been rearranged according to the new classification of Barnett et al., 2000 (Table 3). Table 3. Correspondence between the Lodder taxonomical classification and Barnett taxonomy for yeast biodiversity in Dealu Mare vineyard-valea Calugareasca centre ( ) GENUS Lodder and Kreger van Rij (1967) Saccharomyces Barnett and col. (2000) Saccharomyces SPECIES Lodder and Kreger van Rij (1967) Bayanus exiguus cerevisiae ellipsoideus heterogenicus florentinus fructuum italicus ovifornis steinerii uvarum logos Barnett and col. (2000) bayanus exiguus cerevisiae Torulaspora rosei delbruecki Pichia Pichia membranaefaciens membranifaciens Candida Pichia mycoderma norvegensis Candida peliculosa pelliculosa Torulopsis Candida stellata stellata Schizosaccharomyces Schizosaccharomyces pombe pombe Kloeckera Kloeckera apiculata apiculata Rhodotorula Rhodotorula mucilaginosa mucilaginosa During the non-saccharomyces species at the beginning of the alcoholic fermentation have been 88%, the dominant species being K. apiculata (40%) and Pichia norvegensis (14%). In the middle and at the end phase of alcoholic fermentation the dominant species belong almost to Saccharomyces genus. During our study ( ) a more complex biodiversity has been noticed inside the non-saccharomyces species, especially in the two fermentation phases. In the middle of the fermentation 10 new yeast species with fermentation potential have been isolated. From these species, 50% can be found also at the end of the alcoholic fermentation (Table 4). 118 Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011)

7 Yeast biodiversity evolution over decades in Dealu Mare-Valea Calugareasca vineyard Tabel 4. Comparative analysis of yeast biodiversity in Dealu Mare vineyard-valea Calugareasca Centre between and periods. SPECIA Beginning of fermentation Middle of fermentation End of fermentation Candida colliculosa % - 1.5% Candida famata % % - Candida lusitaniae % - Candida pelliculosa 8.% 2.5% - 2.6% - 1.5% Candida sphaerica % - 1.5% Candida stellata 9.0% 2.5% Candida utilis % - 3.5% - Geotrichum penicillatum % - Kloeckera apiculata 40.0% 5.0% Kloeckera africana 7.0% Clavispora lusitaniae % Pichia ohmeri % - Pichia jadinii % Pichia anomala % Pichia membranifaciens 5.0% % - Pichia norvegensis 14.0% Debaryomyces hansenii % - 4.3% - - Saccharomyces cerevisiae 12.0% 7.5% 98.0% 49.6% 85.0% 77.5% Saccharomyces bayanus % - Saccharomyces exiguus % - 3.0% - Torulaspora delbruecki % 5.2% - 7.5% Rhodotorula glutinis - 2.5% - 1.7% - 3.0% Rhodotorula minuta - 5.0% - 4.3% - - Rhodotorula mucilaginosa 5.0% 10.0% - 6.1% 2.0% - The causes of the increase of the biodiversity complexity during four decades which can be taken into account are linked to the weather changes and to the use of pesticides. Inside the Saccharomyces species also should be taken into account the use of commercial wine yeast strains originated from different vineyards in Europe (Germany, France, etc). Talking about the weather, the global heating may affect the yeast biodiversity. In Dealu Mare region, the medium vegetation temperature has increased in the last four decades with 1.68 o C (from o C to o C) and the vine resources increased with 304 o C. The rainfalls during the vegetation increased too, from 372 mm to 471 mm. The monthly distribution is different in the two studied periods. In the highest rainfall level was measured in the first 3 vegetation months, while in in the second vegetation phase. An important fact is that in our first sampling year, 2007, there have been noticed climacteric anomalies, being the hottest year in the last 107 years (the absolute maximum temperature registered has been 44.3 o C). Consequently, it was a year of drought, similar to Another cause of the biodiversity change should be the nature and the quantity of the used pesticides in the vineyards. It was reported that this chemicals can affect the metabolic activity of yeast, decrease also the yeast biomass production associated with the biodiversity stimulation and kills the fermentative yeasts in the favor of non-fermentative ones. Conclusions During there have been isolated, purified and identified 262 yeast strains from Dealu Mare vineyard- Valea Calugareasca centre. These strains belong both to Ascomycota and Basidiomycota and we have identified 24 species belonging to 12 genera, from which 7 are sporogene. In the years 70 s in the beginning of the fermentation non-saccharomyces species were Kloeckera apiculata (40%) and Pichia norvegensis (14%), while in the last 3 years new species have been identified, as Candida famata (3) and Debaryomyces hansenii (2). Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011) 119

8 MATEI RADOI FLORENTINA, BRINDUSE ELENA, NICOALE GETUTA, TUDORACHE AURELIA, TEODORESCU RAZVAN It has been finally found that in our days there is a higher complexity inside the vineyard yeast biodiversity, especially in the first two alcoholic fermentation phases. During the middle of the fermentation ten new species have been isolated and these represent 50% from the fermentative strains. Some of them are founded also in the ending phase of the process. The increase of the yeast species in the vineyard in the last four decades stands some hypothesis: the increase of the average temperature ( o C) during the vegetative phase in the vineyard, the climacteric anomalies registered on our first study year (2007), the use of pesticides which may reduce the useful yeast biomass in the favor of non-fermentative species. Also, should have been taken into account the commercial yeasts employed in the last decades, isolated from other vineyards, which are less adapted to local conditions. The Saccharomyces sp. purified strains have been tested under laboratory conditions for their fermentative power [Radoi &colab, 1998] and ethanol resistance [Antoce & colab, 2011] and about 30 strains have been kept for further oenological investigations in order to retain the most valuable autochthonous strains for winemaking. Further analyses have been performed (data not published) by the tools of molecular biology (mitochondrial DNA RLFP analysis, PCR amplification), in order to analyze a possible migration of the yeasts and to have an overview of the actual wine yeast biodiversity. Acknowledgments This research has been funded by the national project PNCDI II /2007-DIVINMOL. References 1. ARINA OANA ANTOCE, IOAN C. NAMOLOSANU, FLORENTINA MATEI RADOI (2011): Comparative study regarding the ethanol resistance of some yeast strains isolated from Romanian vineyards. Romanian Biotechnol. Lett., Vol.16, No.1 (in press). 2. BARNETT, J. A., PAYNE, R. W. & YARROW, D. Yeasts: Characteristics and Identification, 3 rd Ed. Cambridge: Cambridge University Press, (2000). 3. BURCEA MIRELA, Cercetari privind variabilitatea naturala si indusa la levuri in vederea detectarii unor suse valoroase pentru vinificatie. Doctoral Thesis, USAMV Bucharest, (1998). 4. BURCEA MIRELA, RADOI, F., Cariotype analysis of some yeast strains isolated from Panciu wine region. Rom. Biotech. Lett., 10 (2), p , (2005). 5. DEAK, T., Simplified techniques for identifying foodborne yeasts. Int J Food Microbiol 19, 15-26, (1993). 6. FLEET, G.H. AND HEARD, G.M. Yeasts: growth during fermentation In: Wine Microbiology and Biotechnology, Harwood Academic Publishers, Chur, Switzerland, pp (1993). 7. KONTEK A., KONTEK ADRIANA, Caracteristicile morfofiziologice si tehnologice ale unei tulpini de Saccharomyces carlsbergensis izolata din vin, Anale ICVV. Vol VI, (1975). 8. KURTZMAN, C. P. & FELL, J. W. The Yeasts - a Taxonomic Study, 4th edn. Amsterdam: Elsevier, (1998). 9. MORTIMER, R., POLSINELLI, M., On the origins of wine yeast, Res. Microbiol. 150, , (1999). 10. NISIOTOU ASPASIA A., GEORGE-JOHN E. NYCHAS, Yeast Populations Residing on Healthy or Botrytis-Infected Grapes from a Vineyard in Attica, Greece, Applied and Environmental Microbiology, p , Vol. 73, No. 8, (2007). 11. PINA CRISTINA, CRISTINA SANTOS, JOSÉ ANTÓNIO COUTO, TIM HOGG, Ethanol tolerance of five non-saccharomyces wine yeasts in comparison with a strain of Saccharomyces cerevisiae influence of different culture conditions, Food Microbiology, Volume 21, Issue 4, Pages , (2004). 12. PRETORIUS I.S., Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16: , (2000). 13. RADOI-MATEI FL., M. BURCEA, E. BRINDUSE, A.KONTEK, Improvement of Romania wines characteristics by using autochthonous yeast starter cultures. Journal of EcoAgriTourism, Proceeding of the International Conference BIOATLAS 2008, Transilvania Univ. Brasov, Romania, p , (2008). 14. SCHULLER DORIT, HUGO ALVES, SYLVIE DEQUIN, MARGARIDA CASAL, Ecological survey of Saccharomyces cerevisiae strains from vineyards in the Vinho Verde Region of Portugal, FEMS Microbiology Ecology 51, ,(2005). 15. ZOHRE D.E, H ERTEN, The influence of Kloeckera apiculata and Candida pulcherrima yeasts on wine fermentation, Process Biochemistry, Volume 38, Issue 3: , (2002). 16. XUFRE A., H. ALBERGARIA, J. INÁCIO, I. SPENCER-MARTINS, F. GÍRIO, Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations, Int. J.of Food Microb., Volume 108, Issue 3: , (2006). 120 Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011)