The impact of media composition and petite ... - Wiley Online Library

Letters in Applied Microbiology 2000, 31, 46ÿ51

The impact of media composition and petite mutation on the longevity of a polyploid brewing yeast strain C.D. Powell1, D.E. Quain2 and K.A. Smart1 1

School of Biological and Molecular Sciences, Oxford Brookes University, Oxford and 2Bass Brewers, Technical Centre, Burton-on-Trent, UK 0014/2000: received 14 January 2000, revised 3 April 2000 and accepted 4 April 2000

Ageing in Saccharomyces cerevisiae is a ®nite phenomenon, determined by replicative, rather than chronological lifespan. Yeast physiological condition is known to in¯uence industrial fermentation performance, however, until recently cellular senescence has not been considered as a brewing yeast stress factor. A polyploid lager yeast (BB11) and a brewery isolate, exhibiting petite mutation were analysed for longevity. It was observed that mitochondrial de®ciency induced a reduction in lifespan. In addition, replicative capacity was perceived to be dependent on environmental conditions.

C . D . P O W E L L , D . E . Q U A I N A N D K . A . S M A R T . 2000.

INTRODUCTION

It has been postulated that lifespan is determined by an organism's genes and in¯uenced by environmental factors (Jazwinski 1990; Sinclair et al. 1998). Analysis of yeast longevity in haploid strains has indicated that the way in which senescence affects individual strains is distinct, leading to the hypothesis that ageing is a strain-speci®c phenomenon (Mortimer and Johnston 1959; Austriaco 1996). Due to stresses imposed on yeast during the brewing process, the frequency of genetic drift and mutations is high. Most of the mutations which occur in brewing yeast result in the loss of a particular function, the most frequent being the loss of respiratory function or `petite' mutation (Stewart 1996). Prolonged storage of production strain populations in holding vessels (Morrison and Suggett 1983), ethanol stress (Bandas and Zakharov 1980; Ibeas and Jimenez 1997) and starvation (Heidenreich and Wintersberger 1998) are known to cause an increase in the occurrence of petite mutations. Petite mutations occur spontaneously in 1±2% of a normal yeast culture (Bernardi 1979), however, Morrison and Suggett (1983) observed that approximately 4% of yeast harvested from fermenters and up to 50% of stored yeast were respiratory de®cient. Physiologically, petites exhibit an altered cell membrane and cell wall morphology (Emandes et al. 1993). Inoculating a fermentation with respiratory-de®cient yeast can result in poor utilization of fermentable sugars (Spencer et al. 1983), inappropriate cell aggregation or ¯ocCorrespondence to: K.A. Smart, School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, OX3 0BP, UK (e-mail: [email protected]).

culation behaviour (Emandes et al. 1993), reduced ethanol production and ¯avour defects (Morrison and Suggett 1983). In addition, the occurrence of non-viable cells increases and the rate of fermentation is reduced (Emandes et al. 1993). It has been suggested that mitochondria play an important role in ageing as they are the major intracellular source of free radicals (Wei et al. 1998) or reactive oxygen species (ROSs), implicated as a contributory factor in senescence (Harman 1956). Of greater signi®cance is the fact that mitochondria are known to be required for resistance to oxidative stress in yeast (Grant et al. 1997) as they supply the energy required to repair damaged material and maintain the cell's somatic function, potentially delaying the onset of the senescent phenotype. In addition to the genetic impact on cellular senescence, investigations into yeast longevity have indicated that lifespan is in¯uenced by growth media and carbon source. It has been observed that substituting ethanol for glucose as a carbon source results in an increased longevity in both diploid (Muller et al. 1980) and haploid (Barker et al. 1999) strains. However, Rodgers et al. (1999) reported that the response of brewing yeast longevity to growth on maltose and ethanol is strain speci®c. The yeast growth and fermentation medium utilized in breweries is known as wort. Despite being brewed to the same formula, different batches of wort frequently exhibit differences in nutrient composition, amino acids and salts. Synthetic media, semide®ned wort substitute (WS) and de®ned wort substitute (DWS) offer reproducible alternatives to brewery wort, mimicking production media in terms of fermentable carbohydrates, amino acids, vitamins and trace elements (Kennedy et al. 1997). It is suggested that media composi= 2000 The Society for Applied Microbiology

LIFESPAN AND BREWING YEAST

tion may in¯uence the longevity of brewing yeast strains. Here we report the impact of petite mutation and media composition on brewing yeast lifespan and senescence. MATERIALS AND METHODS Yeast strains

The yeast strain BB11 and a respiratory-de®cient mutant, designated BB11p, were provided by Bass Brewers (Burton-on-Trent, UK). Media and growth conditions

All media components were supplied by Oxoid (Richmond, CA, USA). Each strain was maintained and propagated on YPD media (2% [w/v] bacteriological peptone, 1% [w/v] yeast extract, 2% [w/v] glucose), 12% agar was added when solid medium was required. Media were sterilized immediately following preparation by autoclaving at 121 C and 1105 Pa for 15 min. Lifespan analysis media. Each strain was examined for longevity using YPD medium. For BB11 three types of additional media were utilized. Wort (1058 gravity) was obtained from Bass Brewers and centrifuged at 4000 rev minÿ1 for 10 mins to remove particulate matter. Wort was sterilized at 121 C and 1105 Pa for 15 min Two types of wort substitute were produced: a semide®ned wort substitute designated WSQ (adapted from Quain and Boulton 1987) (Table 1) and a fully de®ned wort substitute, WSK (Kennedy et al. 1997) (Table 2). WSK media (017% [w/v] Bacto yeast nitrogen base without amino acids and ammonium sulphate (Difco, Detroit, MI, USA) was supplemented with amino acids and ammonium sulphate, pH adjusted to 52 using NaOH and pasteurized at 60 C for 30 min. This was added to a

47

solution of carbohydrates with citric acid, CaSO42H2O and agar (12% [w/v]), previously sterilized at 121 C and 1105 Pa for 15 min. For WSK all carbohydrates were supplied by Oxoid, amino acids were obtained from Sigma Aldrich UK. Determination of cell age

Agar plates (YPD, WSQ, WSK or wort) of no more than 5 mm thickness were inoculated with a single yeast colony and incubated for 48 h at 25 C. The resultant microcolony was then examined using a Zeiss microscope (Jena, Germany) with a long working distance 40 objective lens, by viewing through the Petri dish and agar. Cells were manipulated using a micromanipulation glass needle. Virgin cells were isolated by the manipulation of newly separated buds away from mid-sized mother cells. Careful monitoring of cell cycle progression and subsequent separation of newly generated daughter cells allowed the development from virgin to aged mother cell to be investigated. Plates were incubated at 25 C during the day and at 4 C overnight to decrease growth rate and prevent excessive division. Where necessary, ®lter paper soaked in sterile deionized water was placed in the lid of each Petri dish to prevent desiccation of the medium. For each media type cohorts of cells were monitored using seven replicate plates (10 cells per plate). A total of more than 65 cells were monitored for each strain. Data analysis

The data obtained from micromanipulation were expressed as the mean and maximum10% (mean Hay¯ick limit of the upper decile population) of the lifespan. The standard deviation of each sample group was also calculated. Signi®cance was determined using the heteroscedastic stu-

Table 1 Composition of WSQ (adapted from Quain and Boulton 1987). A salt solution and a carbohydrate solution are prepared

individually and mixed prior to use. Salt solution is prepared by dissolving each salt in turn Component

Final composition (g lÿ1)

Dextrin Maltose Sucrose Fructose Glucose Yeast extract Bacteriological peptone

405 750 225 225 150 100 125

Component


(NH4)2SO4 NaCl CaCl2 MgCl2 KH2PO4 FeCl3

60 25 05 35 50 0.015

= 2000 The Society for Applied Microbiology, Letters in Applied Microbiology, 31, 46ÿ51

48 C . D . P O W E L L E T A L .

Table 2 Composition of WSK (Kennedy et al. 1997). An amino acid solution and a carbohydrate solution are prepared individually and

mixed prior to use. Citric acid and CaSO42H2O are added to the carbohydrate solution before sterilization. Amino acid solution is prepared by dissolving each acid in turn Component


Glucose Fructose Sucrose Maltose Citric acid CaSO42H2O L-Aspartic acid L-Threonine L-Serine L-Asparagine L-Glutamine L-Glutamic acid L-Proline

104 46 35 1155 0625 0215 0.0675 0.0468 0.0375 0.1286 0.0052 0.0778 0.2729

Component Glycine

L-Alanine L-Valine L-Methionine L-Isoleucine L-Leucine L-Tyrosine L-Phenylalanine L-Tryptophane L-Lysine hydrochloride L-Histidine hydrochloride L-Arginine hydrochloride

Ammonium sulphate

dent T-test, and was deemed to have been demonstrated when the value did not exceed 005 (5% con®dence level). RESULTS Brewing yeast senescence is influenced by growth media composition

In order to investigate the relationship between growth media and lifespan, the lager strain BB11 was examined for longevity on YPD, brewery wort (1058 gravity) or one of two forms of wort substitute (WSQ, WSK). BB11 displayed a unique ageing pro®le on each medium (Fig. 1), indicating that for this strain longevity is in¯uenced by media composition.

Final composition (mg lÿ1) 284 884 936 234 496 1218 802 956 423 1122 509 1384 1307

Cells cultivated on brewery wort displayed a mean lifespan of 39 10 and a maximum10% lifespan of 53 2. BB11 cells grown on wort substitutes exhibited a reduced mean lifespan (P < 005) when compared with those investigated for longevity on brewery wort (Table 3). Cells grown on WSQ displayed the shortest lifespan, 22 12 and 45 3 for mean and maximum10%, respectively (Table 3). WSK cells exhibited lifespans which more closely resembled those grown on wort. The mean and maximum10% lifespan of cells propagated on WSK was 31 15 and 52 2, respectively. Cohorts of BB11 cells propagated on YPD also displayed a signi®cant decrease in mean lifespan when compared with those grown on wort (P < 005) (Table 3). However, when the longevity of BB11 was compared on YPD and WSK, no signi®cant difference was observed (P > 005). Interestingly, despite variation in mean lifespan, the maximum10% lifespans of cells grown on YPD, wort and WSK did not differ signi®cantly from one another (P < 005).

The impact of petite mutation on brewing yeast longevity

Fig. 1 Mortality pro®les of the lager strain BB11. (&) YPD,

(W) WSQ, (~) WSK, (^) wort

Analysis of haploid strains has indicated that the impact of petite mutation on longevity may be strain speci®c (Kirchman et al. 1999), however, there has been no previous analysis of lifespan in respiratory-de®cient brewing yeast. The majority of petite strains analysed to date have exhibited a reduced lifespan. Berger and Yaffe (1998) identi®ed a petite mutant of the strain MYY636 which dis-



49

Table 3 Longevity of the lager strain BB11 on laboratory and industrial growth media

Media type

Cells manipulated

Mean lifespan S.D.

Maximum10% lifespan S.D.

YPD WSQ WSK Wort 1058

215 73 73 71

33 11 22 12 31 15 39 10

51 4 45 3 52 2 53 2

Table 4 Longevity of BB11 and petite mutant (BB11p)

Strain

Cells manipulated

Mean lifespan S.D.

Maximum10% lifespan S.D.

BB11 BB11p

215 71

33 11 19 12

51 4 39 4

played a 40% reduction in lifespan. Studies of the haploid strain CY4 and its petite CY4p within our laboratory have also established the occurrence of a 25% reduction in lifespan (Van Zandycke, Personal Communication). In order to study the impact of petite mutation on brewing yeast longevity, the lifespan of a respiratory-de®cient mutant, BB11p, derived from BB11 was investigated on YPD. When compared with BB11, the petite strain displayed a reduced lifespan (P < 005) (Table 4, Fig. 2). BB11p exhibited a mean lifespan of 19 8 which corresponds to a 40% reduction in lifespan when compared with the original BB11 strain (32 10).

Fig. 2 Mortality pro®les of BB11 and petite (BB11p) analysed for

longevity on YPD. (&) BB11, (W) BB11p

DISCUSSION

It has previously been demonstrated that brewing yeast longevity is in¯uenced by carbohydrate composition (Barker et al. 1999; Rodgers et al. 1999). However, to date there has been no reported comparison of yeast cellular senescence on laboratory and industrial media. Lifespan analysis of the brewing strain BB11 was performed using YPD, brewery wort (1058 gravity), semi-de®ned wort substitute and de®ned wort substitute. It was observed that yeast propagated on wort displayed a greater mean lifespan than on traditional laboratory YPD media. However, the maximum10% lifespan was not signi®cantly different. Despite this observation, it cannot be suggested that growth on wort would consistently result in an extended mean lifespan, because considerable variability in wort composition may occur (Kennedy et al. 1997). A medium of a consistent nutrient composition, re¯ecting the carbon and nitrogen sources typically present in brewery wort was utilized for lifespan determinations. Cells grown on WSQ were observed to display a reduced lifespan when compared with YPD and wort. However, cohorts of cells grown on WSK exhibited a lifespan which more closely resembled that of those propagated on wort. Statistical analysis of the maximum10% lifespan of BB11 revealed there to be no difference between wort and WSK. In addition, WSK grown cell populations were not observed to differ signi®cantly from those grown on YPD in terms of mean or maximum10% lifespan potential. It is postulated that while neither of these media is identical in composition to


50 C . D . P O W E L L E T A L .

wort, utilizing either for lifespan analysis provides a suitable alternative laboratory medium to brewery wort. It was observed that cells propagated on YPD and WSK displayed similar maximum10% lifespans to those grown on wort, despite having a reduced mean lifespan. This indicates that each cell within a population has the potential to survive to a predetermined age regardless of media composition. For those individuals which do not reach this upper limit of longevity, lifespan is reduced by environmental conditions comprising physiological stress, nutritional imbalance or ineffective repair of cellular damage. It is postulated that growth on WSQ may result in impaired lifespan potential due to nutritional imbalance. The petite mutant, BB11p was observed to display a 40% reduction in lifespan compared with that of the wild type. This result supports the hypothesis that mitochondrial function is required in order to maintain longevity in yeast (Coates et al. 1997). As mitochondria are the main source of ROS it has been suggested that a strain with defective mitochondria would produce less free radicals, leading to less-stressed individuals (Guidot et al. 1993; Longo et al. 1996). A decrease in damage accumulation would in turn lead to enhanced longevity (Miguel and Fleming 1996; Wei et al. 1998). However, as mitochondria play such an important role in producing ATP it has also been postulated (Grant et al. 1997) that mitochondrial function is essential for resistance to oxidative stress. This is likely to be due to the energy-requiring process needed for ROS detoxi®cation or repair of damaged molecules. This theory has yet to be speci®cally applied to yeast ageing, although this seems the most likely explanation for the reduced lifespan observed in the petite mutant of BB11. Given that respiratory-de®cient yeasts do not perform well in brewery fermentations when compared with their `grande' counterparts, it is postulated that the altered ageing pro®le observed in petite mutants may be a signi®cant causative factor in poor fermentation performance.

ACKNOWLEDGEMENTS

Christopher Powell is supported by a BBSRC Industrial CASE studentship with Bass Brewers. The authors are grateful to the Directors of Bass Brewers for permission to publish this work.

REFERENCES Austriaco, N.R. (1996) Review: to bud until death: the genetics of ageing in the yeast. Saccharomyces. Yeast 12, 623±630. Bandas, E.L. and Zakharov, I.E. (1980) Induction of rho± mutations in yeast Saccharomyces cerevisiae by ethanol. Mutation Research. 71, 193±199.

Barker, M.G., Brimage, L.J.E. and Smart, K.A. (1999) Effect of Cu, Zn superoxide dismutase disruption mutation on replicative senescence in Saccharomyces cerevisiae. FEMS Microbiology Letters 177, 199±204. Berger, K.B. and Yaffe, M.P. (1998) Prohibitin family members interact genetically with mitochondrial inheritance components in Saccharomyces cerevisiae. Molecular and Cellular Biology 18, 4043±4052. Bernardi, G. (1979) The petite mutation in yeast. Trends in Biochemical Sciences 4, 197±201. Coates, P.J., Jamieson, D.J., Smart, K.A. and Hall, P.A. (1997) The prohibitin family of mitochondrial proteins regulate replicative lifespan. Current Biology 7, R607±R610. Emandes, J.R., Williams, J.W., Russell, I. and Stewart, G.G. (1993) Respiratory de®ciency in brewing yeast strains ± effects on fermentation, ¯occulation and beer ¯avour components. Journal of the American Society of Brewing Chemists 51, 16±20. Grant, C.M., MacIver, F.H. and Dawes, I.W. (1997) Mitochondrial function is required for resistance to oxidative stress in the yeast. Saccharomyces cerevisiae. FEBS Letters 410, 219±222. Guidot, D.M., McCord, J.M., Wright, R.M. and Repine, J.E. (1993) Absence of electron transport (Rhoo state) restores growth of a manganese-superoxide dismutase-de®cient Saccharomyces cerevisiae in hyperoxia. Journal of Biological Chemistry 268, 26699±26703. Harman, D. (1956) Ageing: a theory based on free radical and radiation chemistry. Journal of Gerontology 114, 298±300. Heidenreich, E. and Wintersberger, U. (1998) Replication-dependent and selection-induced mutations in respiration-competent and respiration-de®cient strains of Saccharomyces cerevisiae. Molecular and General Genetics 260, 395±400. Ibeas, J.I. and Jimenez, J. (1997) Mitochondrial DNA loss caused by ethanol in Saccharomyces ¯our yeasts. Applied and Environmental Microbiology 63, 7±12. Jazwinski, S.M. (1990) Aging and senescence of the budding yeast Saccharomyces cerevisiae. Molecular Microbiology 4, 337±343. Kennedy, A.I., Taidi, B., Dola, J.L. and Hodsgon, J.A. (1997) Optimisation of a fully de®ned medium for yeast fermentation studies. Food Technology and Biotechnology 35, 261±265. Kirchman, P.A., Kim, S., Chi-Yung, L. and Jazwinski, S.M. (1999) Interorganelle signalling is a determinant of longevity in Saccharomyces cerevisiae. Genetics 152, 179±190. Longo, V.D., Gralla, E.B. and Valentine, J.S. (1996) Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae. Journal of Biological Chemistry 271, 12275±12280. Miguel, J. and Fleming, J. (1996) Theoretical and experimental support for an oxygen radical-mitochondrial injury hypothesis of cell aging. In Free Radicals, Aging and Degenerative Diseases ed. Johnson, J.E., Harman, D., Walford, R., Miguel, J. pp. 51± 74. New York, NY: Liss. Morrison, K.B. and Suggett, A. (1983) Yeast handling, petite mutants and lager ¯avour. Journal of the Institute of Brewing 89, 141±142. Mortimer, R.K. and Johnston, J.R. (1959) Life span of individual yeast cells. Nature 183, 1751±1752.



Muller, I., Zimmermann, M., Becker, D. and Flomer, M. (1980) Calender lifespan versus budding lifespan of Saccharomyces cerevisiae. Mechanisms of Aging and Development 12, 47±52. Quain, D.E. and Boulton, C.A. (1987) The propogation of yeast on oxidative carbon sources ± advantages and implications. Proceedings of the EBC Congress 21, 417±424. Rodgers, D.L., Kennedy, A., Hodgson, J.A. and Smart, K.A. (1999) Lager brewing yeast ageing and stress tolerance. Proceedings of the EBC Congress 27, 671±679. Sinclair, D.A., Mills, K. and Guarente, L. (1998) Aging in Saccharomyces cerevisiae. Annual Review of Microbiology 52, 533±560.

51

Spencer, J.F.T., Spencer, D.M. and Miller, R. (1983) Inability of petite mutants of industrial yeasts to utilize various sugars and a comparison with the ability of the parent strains to ferment the same sugars microaerophilically. Zeitcschrift Fur Naturforschung C-A Journal of Biosciences 38c, 405±407. Stewart, G. (1996) Yeast performance and management. Brewer 82, 211±215. Wei, Y.H., Pang, C.Y., Lee, H.C. and Lu, C.Y. (1998) Roles of mitochondrial DNA mutation and oxidative damage in human ageing. Current Science 74, 887±893.
