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Growth of non-Saccharomyces yeasts affects nutrient availability for Saccharomycescerevisiae during wine fermentation

Karina Medina a Eduardo Boido a Eduardo Dellacassa b Francisco Carrau a

a Seccioacuten Enologiacutea Departamento de Alimentos Facultad de Quiacutemica Universidad de la Repuacuteblica Montevideo 11800 Uruguayb Caacutetedra de Farmacognosia y Productos Naturales Departamento de Quiacutemica Orgaacutenica Facultad de Quiacutemica Universidad de la Repuacuteblica Montevideo 11800 Uruguay

a b s t r a c ta r t i c l e i n f o

Article history

Received 4 February 2012Received in revised form 30 April 2012Accepted 11 May 2012Available online 16 May 2012

Keywords

Non-Saccharomyces

Sluggish wine fermentationAssimilable nitrogenVitamins

Yeast produces numerous secondary metabolites during fermentation that impact 1047297nal wine qualityAlthough it is widely recognized that growth of diverse non-Saccharomyces (NS) yeast can positively affect1047298avor complexity during Saccharomyces cerevisiae wine fermentation the inability to control spontaneousor co-fermentation processes by NS yeast has restricted their use in winemaking We selected two NS yeastsfrom our Uruguayan native collection to study NSndashS cerevisiae interactions during wine fermentation Theselected strains of Hanseniaspora vineae and Metschnikowia pulcherrima had different yeast assimilable nitro-gen consumption pro1047297les and had different effects on S cerevisiae fermentation and growth kinetics Studiesin which we varied inoculum size and using either simultaneous or sequential inoculation of NS yeast andS cerevisiae suggested that competition for nutrients had a signi1047297cant effect on fermentation kineticsSluggish fermentations were more pronounced when S cerevisiae was inoculated 24 h after the initial stageof fermentation with a NS strain compared to co-inoculation Monitoring strain populations using differentialWL nutrient agar medium and fermentation kinetics of mixed cultures allowed for a better understanding of strain interactions and nutrient addition effects Limitation of nutrient availability for S cerevisiae was shownto result in stuck fermentations as well as to reduce sensory desirability of the resulting wine Addition of diammonium phosphate (DAP) and a vitamin mix to a de1047297ned medium allowed for a comparison of nutrientcompetition between strains Addition of DAP and the vitamin mix was most effective in preventing stuck

fermentations copy 2012 Elsevier BV All rights reserved

1 Introduction

Indigenous yeasts present in grape musts at the onset of wine fer-mentation can be divided broadly into two groups ie the wine yeastSaccharomyces cerevisiae and the non-Saccharomyces (NS) yeasts Anumber of different commercial S cerevisiae strains have been devel-oped for winemaking that can establish dominant populations fromthe beginning of fermentation so as to ensure complete consumptionof sugars Although a large population of active yeast cells is typicallyused for inoculation many studies have shown that indigenousstrains are not completely suppressed and can develop to a signi1047297-cant extent during early stages of juice fermentation (Ciani et al2010 Fleet 2008) Indigenous NS yeasts are found predominantlyon grapes and to lesser extent on cellar equipment (Zott et al2008) NS yeasts are present in highest numbers in grape must priorinoculation with commercial S cerevisiae In what is termed ldquosponta-neous fermentationrdquo there is a sequence of dominance by various NS

grape must yeasts followed by S cerevisiae which can then completefermentation (Fleet 2003) Indigenous yeasts have been reported tocontribute either positively or negatively to overall sensory charac-teristicsof wine Such results could be explained in part by the diver-sity of NS yeasts in grape must and the limited number of studies indifferent laboratories with consistent methodologies for controllingavailable nutrients (Carrau 2003) Several studies have shown thatNS yeast strains can be detected throughout fermentation ( Jolly etal 2006) and that their dominance during early stages can in1047298uencethe 1047297nal composition of wine (Romano et al 1997) However a num-ber of studies have shown that the presence of NS yeast is associatedwith increased wine quality and complexity (Anfang et al 2009Carrau 2003 Ciani et al 2006 Egli et al 1998 Fleet 2003Henick-Kling et al 1998 Jolly et al 2003a Peacuterez et al 2011 ) Eventhough S cerevisiae is responsible for a majority of the ethanol inwine the presence of NS yeasts may have a signi1047297cant effect on theproduction of aroma compounds including esters higher alcoholsacids and monoterpenes (Swiegers et al 2005)

The main NS yeasts present on grape and in grape must are apiculateand include Kloeckera apiculataHanseniaspora uvarum K apisH

guillermondii K africanaH vineae K corticisH osmophila and K

International Journal of Food Microbiology 157 (2012) 245ndash250

Corresponding author at Facultad de Quiacutemica Universidad de la Repuacuteblica Monte-video 11800 Uruguay Tel +598 29248194 fax +598 29241906

E-mail address fcarraufqeduuy (F Carrau)

0168-1605$ ndash see front matter copy 2012 Elsevier BV All rights reserved

doi101016jijfoodmicro201205012

Contents lists available at SciVerse ScienceDirect

International Journal of Food Microbiology

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e i j f o o d m i c r o



javanicaH occidentalis ( Jolly et al 2006) These yeasts generally havemoderate fermentative capacity and are important in the production of volatile aroma compounds (Viana et al 2011) There is concern aboutthe use of mixed cultures of NS apiculates and Saccharomyces in that theinitial rapid growth of some NS strains may have an inhibitory effect onsubsequent growth of S cerevisiae This phenomenoncould be implicatedin causing sluggish or stuck fermentations Nutrient limitation effects onmixed cultures wine fermentation have generally not been studied how-

ever some vitamins such asthiamine andpantothenic acid were associat-ed with sluggish wine fermentations (Bataillon et al 1996 Guzzon et al2011 Wang et al 2003) Therefore a very cautionary approach is cur-rently taken when considering use of NS apiculate strains in wineproduction

Metschnikowia pulcherrima can occur in high numbers in grapemust ( Jolly et al 2003b Peacuterez et al 2011 Schutz and Gafner1993) and are known to produce high concentrations of esters(Bisson and Kunkee 1991) especially the pear-associated esterethyl caprylate (Clemente-Jimenez et al 2005 Lambrechts andPretorius 2000) In addition production of undesirable volatilecompounds was not detected during mixed culture fermentationsusing M pulcherrima and S cerevisiae (Zohre and Erten 2002) anda positive sensory contribution to wine was shown for Chenin blancin sequential fermentations ( Jolly et al 2003b) However utilizingthe same treatment wines of Chardonnay were judged to be of an in-ferior quality compared with fermentation using only S cerevisiae

( Jolly et al 2003a)The aim of this study was to evaluate the effect of nutrient depletion

in a fermentation broth by NS strains during growth and fermentationin mixed cultures with S cerevisiae It is proposed that competition fornutrients is themain cause forinconsistentresults in NSndashSaccharomyces

co-fermentations We also suggest that understandingnitrogen balanceand vitamin requirements for the Saccharomyces inoculant could allowfor scaling-up the use of NS strains to the production level inwinemaking by helping to prevent sluggish or stuck fermentationsand improving the aroma composition and general positive attributesof the 1047297nal wine (Carrau 2003 Ciani et al 2010 Fleet 2008) Over500 strains of NS yeast were isolated from the Uruguayan winemaking

environment over a 5‐year period More than 20 of these NS strainsfrom grapesand theinitialstages of fermentation were previously iden-ti1047297ed that positively contributed to aroma and quality of white and redwines ( Jubany et al 2008 Peacuterez et al 2011) From this collection weselected two strains of the two main species present H vineae andM pulcherrima in order to study their potential application in simulta-neous or sequential fermentation with a conventional Saccharomyces

wine strain

2 Materials and methods

21 Yeast strains

The S cerevisiae strain used was ALG804 (DSM Denmark) a strainthat is used in commercial wine production NS strains used were H

vineae T025AF (Hv) isolated from wine fermentation of Tannat andM pulcherrima M0326 G (Mp) isolated from grape must of Merlotand both strains were previously characterized genetically (Barquetet al 2012)

Inocula were prepared in the same fermentation medium by incu-bation for 12 h on a rotary shaker at 150 rpm and 25 degC Inoculumsize was 5times105 cellsmL of medium except those indicated at 5times106

cellsmL Cultures were maintained at 4 degC on YEPD medium slants(1 yeast extract 2 peptone 2 glucose 2 agar containing 01 Mcitrate-phosphate buffer pH 45) Antagonistic or killer effect betweenstrains was analyzed using YEPD plating medium for killer neutral andsensitive determinations (Carrau et al 1993) resulting in a neutral

phenotype between these strains

22 Fermentation conditions

Yeast assimilable nitrogen (YAN) consumption pro1047297les for NSstrains were determined during fermentation of a Muscat of Alexan-dria white grape must containing 178 mg NL YAN 210 gL totalsugars and pH 35 The medium was 1047297lter sterilized using 045 μ m(Sartorius) and fermented in duplicate at 20 degC Static 125 mL batchgrape juice fermentations were done in 250 mL Erlenmeyer 1047298asks

(sealed with Muller valves 1047297

lled with pure sulfuric acid)The effects of nutrient availability during mixed culture fermenta-tions were studied using a previously described but modi1047297ed de1047297nedfermentation medium based on the nutrient composition of grape

juice (Carrau et al 2005) The modi1047297ed de1047297ned medium had abasic total nitrogen content of 50 mg NL with each amino acid andammonium component added in the same proportions previously in-dicated (Carrau et al 2005) The 1047297nal YAN concentration of 125 mg NL was made increasing the basic concentration by supple-mentation with diammonium phosphate (DAP) This YAN amountwas not a limiting concentration for complete fermentation of sugarsby strain ALG804 Equimolar concentrations of glucose and fructosewere added to 200 gL Addition of 300 mgL of DAP was done tofermentations in experiments where indicated in day three afterinoculation obtaining a 1047297nal YAN concentration of 188 mgNL inthese treatments Vitamin solutions were prepared and added to thefollowing 1047297nal concentrations (mgL) myo-inositol 100 pyridoxineHCl 2 nicotinic acid 2 calcium pantothenate 1 thiamine HCl 05PABA 02 Ribo1047298avine 02 biotin 0125 Folic acid 02 Thiamine andcalcium pantothenate were excluded from the vitamin solution whereindicatedErgosterol wasadded to a 1047297nal concentration of 10 mgL pre-viously solved in ethanol as described previously (Henschke and

Jiranek 1993) The 1047297nal pH of the medium was adjusted to 35 withHCl The medium was 1047297lter sterilized using 022 μ m (type Millipore)Static 125 mL batch fermentations were done in 250 mL Erlenmeyer1047298asks (sealed with Muller valves 1047297lled with pure sulfuric acid) at20 degC in duplicate to simulate wine making conditions Fermentationactivity was measured by CO2 weight loss expressed in grams per100 mL and decreasing YAN levels using the formaldehyde method

(Zoecklein et al 1995) Samples were taken once a day to measurecell growthin an improved Neubauer chamberRelative yeast cell num-bers distributions were determined during growth or fermentationusing the differential culture growth WL nutrient medium (Medinaet al 1997 Pallmann et al 2001) In this medium H vineae growthis identi1047297ed by its intense brilliant green color whereas M pulcherrima

acquires a light green and strain ALG804 a matt clear creamy color

23 Statistical analysis

ANOVA was done to establish signi1047297cance of differences for strainsinoculated in fermentation performance growth YAN consumptionand nutrient addition effects Statistica 71 software was used foranalysis

3 Results

31 YAN consumption in NS strains

YAN consumption pro1047297les for strains Hv Mp and ALG804 areshown in Fig 1A The YAN was consumed rapidly and completely inthree days by the Mp and ALG804 strains whereas YAN consumptionremained at ca 90 after this period for the Hv strain Fermentationand growth pro1047297les for the strains during YAN consumption is alsoshown in Fig 1B and C Although strain Hv had a higher fermentationrate than strain Mp YAN consumption was slower than that of strainMp The faster growth rate of strain Mp was consistent with its higherYAN consumption A reduced fermentation pro1047297le was seen for strain

Mp relative to that for strain Hv and both NS yeasts had only moderate

246 K Medina et al International Journal of Food Microbiology 157 (2012) 245ndash 250



fermentation capacity compared with strain ALG804 Similar resultswere obtained using the chemical de1047297ned medium utilized further onin controlled mixed cultures

32 Sluggish and stuck fermentation in mixed cultures

Mixed culture fermentation of a de1047297ned grape juice medium usingstrain ALG804 and Hv or ALG804 and Mp resulted in sluggish(ALG804Hv) or stuck (ALG804Mp) fermentation compared withfermentations using pure cultures of ALG804 (Fig 2) This behaviorcould be explained by competition for nutrients or other cellndashcell in-teraction effects Regarding cellndashcell interactions no antagonistic or

yeast killer effect was found between the NS strains and strain

AL804 using the described killer interaction method (data notshown) Co-inoculation of strain AL804 with strain Mp resulted in a

greater inhibition in mixed fermentation compared with co-inoculation with strain Hv This result is consistent with the relativelyhigher growth rate and consumption of YAN by strain Mp seen inFig 1

The aroma of individual strain fermentations was consideredpleasant fruity and complex in agreement with the studies in whichthese native yeasts were selected while when inoculated in mixedcultures in the same grape must some undesirable notes were indi-cated by the tasters (Carrau 2003) We thus proposed that limitationof nutrient availability in the medium due to growth of a NS strainresulted in strain AL804 having sluggish or stuck fermentation andin part explaining the production of some off 1047298avor aroma com-pounds The effects of the co-inoculated NS strains on the fermenta-tion capacity of strain AL804 were further investigated by altering

the size of inoculum timing of inoculation of strain AL804 and nutri-ent supplementation

33 Effect of inoculum size and timing of inoculation on population com-

position in mixed cultures

We used a simple differential agar medium (WL nutrient medi-um) to monitor relative changes in populations of strains ALG804and Hv in mixed cultures after inoculation Inoculation of equal num-bers of each strain (5times 105 cellsmL) at the beginning of the fermen-tation allowed for strain ALG804 to eventually dominate the cultureafter six days (see Fig 3A) When the relative number of Hv cellsused for inoculation was increased tenfold (5 times106 cellsmL) strainALG804 was able to grow to ca 35 of the culture after six days

(Fig 3A) The higher proportion of Hv cells at the beginning of the

0

05

1

15

2

0 1 2 3

g 1 0 0 m L C O

2

days

Fermentation

Mp

Hv

ALG 804

0

20

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0 1 2 3

x 1 0 6 c e l l s m L

days

Growth

Mp

HvALG 804

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0 1 2 3days

YAN Consumption

Mp

Hv

ALG 804

mg NLA

B

C

Fig 1 Comparison of YAN consumption (A) fermentation kinetics (B) and growth rate(C) of NS strains Mp and Hv and strain ALG804 during fermentation of Muscat of Alex-

andria grape juice (see Section 2) Results are the average and bars indicate the SD

0

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g

C O 2 1 0 0 m L

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0 2 3 4 5 6 7 8 9 10

g C O 2 1 0 0 m L

Days

ALG804Mp

Mp+ALG804

ALG804Hv

Hv+ALG804

A

B

Fig 2 Fermentation of a de1047297ned grape must containing 125 mgNL at 20 degC using pureor mixed cultures of strain ALG804 and NS strains (A) Inoculation with strains Mp andALG804 and (B) inoculation with strains Hv and ALG804 Results are the average andbars indicate the SD

247K Medina et al International Journal of Food Microbiology 157 (2012) 245ndash 250



fermentation (101 proportion for strains HvALG804) enabled strainHv to use suf 1047297cient available nutrients in the medium so as to effec-tively compromise growth of strain ALG804 To further test if nutrientcompetition between strains restricted growth of strain ALG804 thetiming of inoculation of this strain into the fermentation mediumwas modi1047297ed Fig 3 shows the proportion of strain ALG804 cells pre-sent when strains ALG804 and Hv were inoculated at the same time(Fig 3A) and when strain AL804 was inoculated 24 h after strain Hv(Fig 3B) Comparison of these two pro1047297les shows that the percentageof strain ALG804 cells is higher when strains were inoculated at thesame time When strain ALG804 was inoculated to the fermentationmedium 24 h after inoculation of strain Hv its ability to dominatethe culture after six days was reduced 50 (Fig 3B) Likewise when

inoculation of strain Hv was increased tenfold 24 h prior to inocula-tion with strain ALG804 its ability to dominate the culture was alsoreduced by about 50 (ie from 35 to 15) These results suggestedthat strain Hv consumed nutrients in the medium during the 1047297rst24 h that reduced the ability of strain ALG804 to grow after it was in-oculated into the partially spent medium As shown in Fig 1 strain Hvconsumed more than 50 of YAN in the medium within the 1047297rst 24 hConsistent with this addition of suf 1047297cient YAN at the time of inocula-tion of strain ALG804 24 h after inoculation of strain Hv nearly re-stored the ability of strain ALG804 to fully dominate the culturemedium (Fig 3B) Similar results were obtained with mixed culturesof strain Mp and ALG804 involving increases in size of inoculum ortiming of inoculation (data not shown) In both cases increasing theinoculum to 5times106 cellsmL for the NS strain resulted in a decrease

in fermentation rate for the mixed culture

34 Nitrogen and vitamins in mixed cultures fermentation and growth

Results suggested a competition for nutrients such as YAN inmixed cultures of strain ALG804 and NS strains Further fermentationexperiments with de1047297ned grape must were done to more accuratelyde1047297ne nutrient limitations involving nitrogen as well as vitamins inthe medium Fig 4 shows the results obtained for the two combina-tions of mixed cultures strains MpALG804 and HvALG804 Interest-

ingly we obtained signi1047297

cantly different results depending on the NSstrain Fermentation in mixed cultures of strains Mp and ALG804 wasimproved with the addition of assimilable nitrogen and further im-proved with addition of both nitrogen and a vitamin mixture(Fig 4A) Fermentation in mixed cultures of strains ALG804 and Hvwas not improved with the addition of assimilable nitrogen aloneHowever addition of nitrogen together with vitamins resulted in im-proved fermentation for this mixed culture These results suggestedthat vitamin depletion by strain Hv in mixed cultures with strainALG804 could be a limiting factor in fermentation

In summary nutrient addition on the third day enhanced fermen-tation performance in a signi1047297cant way during mixed culturesAlthough a clear effect on total yeast growth of both strains was notevident (data not shown) the effect of an increase in the proportionof strain ALG804 was clear in the mixed culture as shown in Fig 3that could explain the increase fermentation performance

Vitamin limitation related to sluggish wine fermentations wasreported in two studies one with thiamine (Bataillon et al 1996)and the other with pantothenic acid (Wang et al 2003) The de1047297ned

9930

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S a c A L G 8 0 4

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ALG 804Hv ALG 804 T=24 hours

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S a c A L G 8 0 4

Days

ALG 804Hv ALG 804 T=0

ALG804+Hv 5x10 5

ALG804+Hv 5x10 6

Hv 5x10 5 +ALG804

Hv 5x10 6 +ALG804

Hv 5x10 5 +ALG804+YAN

B

A

Fig 3 Percentage of strain ALG804 colony forming units (cfu) in populations of ALG804ndashHv mixed cultures using inocula for strain Hv of 5times105 or 5times106 cellmLThe two strains were either co-inoculated together (A) or strain ALG804 was inoculat-ed 24 h after inoculation of strain Hv (B) Colony formed units (cfu) was determinedmeasuring the colony color differences in WL nutrient medium during the fermenta-tion process of the similar grape must medium at 20 degC Results are the average andbars indicate the SD

0

1

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g C

O 2 1 0 0 m L

Days

Mp+ALG804

Mp+ALG804+NH4

Mp+ALG804+NH4+Vit

0

1

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g C O 2 1 0

0 m L

Days

Hv+ALG804

Hv+ALG804+NH4

Hv+ALG804+NH4+Vit

A

B

Fig 4 Fermentation curves of mixed cultures of strains ALG804 and either strainMp (A) or strain Hv (B) in which strain ALG804 was inoculated 24 h after addition of the NS strain to a de1047297ned grape must medium Supplementation of the medium withassimilable nitrogen or assimilable nitrogenvitamins was done on day three Fermenta-tions were carried out at 20 degC results are the average of duplicates and bars indicate

the SD




grape must medium could be modi1047297ed so as to exclude these vita-mins Although both these vitamins were previously shown to bekey factors for fermentation performance in S cerevisiae excludingthiamine and pantothenic acid from our vitamin mix had no effecton the ability of the vitamin mix to enhance fermentation (seeFig 5) No signi1047297cant differences were found if thiamine and pan-tothenic acid were excluded from the vitamin mixture comparedwith the complete mixture These results suggest that other vitaminsmight contribute to the fermentation process or that there is a syner-gistic effect between assimilable nitrogen and vitamin mix in oursystem

4 Discussion

Mixed culture fermentation using S cerevisiae and NS grape mustyeasts holds promise as a way to improve wine complexity howeverinteractions between strains and methods of inoculation need to beexamined in more detail (Ciani and Comitini 2011) Previous studies

with mixed cultures suggested that growth of NS strains resulted indepletion of nutrients such as YAN and a poor medium for growthof Saccharomyces strains sluggish or stuck fermentations (Bataillonet al 1996 Fleet and Heard 1993) and loss of desired sensory fea-tures in wines (Carrau 2003 Wang et al 2003) However speci1047297cstudies to clearly demonstrate the importance of nutrient competi-tion related to nitrogen limitation in these mixed cultures have notbeen reported Although studies on nitrogen consumption rate differ-ences between different strains used in mixed cultures with Saccharo-

myces have been limited (Medina et al 1997) it was suggested thatcommercial (Saccharomyces) strains had a relatively increased de-mand for nitrogen compared with selected native strains (Carrau etal 2008) This was recently examined in greater detail in which dif-ferences in amino acid uptake were analyzed during wine fermenta-

tion (Barrajoacuten-Simancas et al 2011)In this study YAN consumption pro1047297le of two NS yeast species M

pulcherrima and H vineae was investigated to study the impact on S

cerevisiae fermentation capacity in mixed cultures The two NSstrains Hv and Mp both consumed nitrogen rapidly with Mp alsohaving a faster growth rate and lower fermentation capacity thatmay explain a higher consumption of YAN Studies with some strainsof S cerevisiae suggested that fermentation was more nitrogen-demanding than that for growth or respiration processes (Backhuset al 2001) whereas Mp showed the opposite behavior This mightbe expected since NS strains may be more adapted to fruit surface en-vironments that may not require signi1047297cant fermentation abilities

Mixed cultures with these NS strains and strain ALG804 showedthat sluggish fermentations were related to nutrient limitation and

growth of a NS strain with Mp having a more pronounced effect

Our results are in agreement with related studies on mixed culturefermentation kinetics of other NSndashSaccharomyces strain combinations(Andorragrave et al 2010) where an increase of YAN consumption wasfound Our results also showed that sluggish fermentations wereslower when inoculation of strain ALG804 was sequential ratherthan inoculated at the same time as the NS strain (simultaneous inoc-ulations) In sequential inoculations the NS strains consumed nutri-ents for growth reducing the ability of the Saccharomyces strain to

grow after it was inoculated whereas when simultaneously inoculat-ed the Saccharomyces strain could negatively affect NS strain growthIn addition slower fermentations were evident when a tenfold in-crease in the NS strain was used for inoculation also suggesting thatnutrient competition effects are a main cause for sluggish or stuck fer-mentations in these situations In our model system using a de1047297nedmedium based on a white grape must the moderate YAN concentra-tion (125ndash178 mg NL) used could explain why we saw a clear effectas nutrients were fully consumed during competition betweenstrains and could indicate nitrogen limitation for both strains in themixed culture Some potentially contradictory results previouslyreported using NSndashSaccharomyces mixed culture investigating slug-gishstuck fermentation or aroma balance in the 1047297nal wine (Ciani etal 2010) might be attributed to YAN levels in the media whichwere either not reported (Clemente-Jimenez et al 2005 Soden etal 2000) or in which concentrations of YAN (or DAP) were too high(over 200 mgNL) (Anfang et al 2009 Egli et al 1998 Viana et al2011 Zott et al 2008 2011) Interestingly it was recently demon-strated that addition of a mixture of 20 amino-acids or a yeast extractgreatly improved the fermentation ef 1047297ciency of K africana (the im-perfect state of H vineae) compared with tequila fermentations sup-plemented with ammonium sulfate alone (Diacuteaz-Montantildeo et al2010 Valle-Rodriacuteguez et al 2012)

Literature in reference to the vitamin suf 1047297ciency effects for growthand fermentation performance by wine yeast is very limited As notedabove only two studies have been reported and these investigatedthe importance of pantothenic acid and thiamine in S cerevisiae

wine fermentation (Bataillon et al 1996 Wang et al 2003) In onestudy it was demonstrated that K apiculata was very ef 1047297cient at strip-

ping thiamine from grape juice in a matter of hours and thus leadingto a de1047297ciency growth of Saccharomyces and a likely cause of stuckfermentations (Bataillon et al 1996) Interestingly studies on thia-mine assimilation during the 1047297rst hours of fermentation showedthat this apiculate strain (K apiculata H uvarum) removed this vita-min from the medium more rapidly than did S cerevisiae and at a sig-ni1047297cant faster rate when low numbers of cells of both strains wereinoculated in mixed cultures (ie 104 cellmL) (Bataillon et al1996) Here we show that assimilable nitrogen together with a vita-min mix signi1047297cantly affected fermentation performance of themixed cultures Furthermore signi1047297cant differences were also foundin the proportions of Saccharomyces and NS strains where an increasein percentage of the Saccharomyces strain was found after nutrientaddition Interestingly no signi1047297cant difference in response was

found between YANndashvitamin addition together versus addition of YANndashvitamin with thiamine and pantothenic acid excluded fromthe mix Indeed the present study suggests that neither thiaminenor pantothenic acid limitation is likely to be responsible for slug-gishstuck fermentations for the strains tested Paradoxically it wasshown that a negative effect of these two vitamins on fermentationrate andor formation of H2S was augmented with YAN contents of over 200 mgNL (Bataillon et al 1996 Wang et al 2003) In that125 mgNL YAN was used in our experimental system perhaps thisnegative effect on fermentation was not relevant In another study itwas suggested that biotin affected fermentation in that a reductionof H2S evolution under YAN de1047297ciency was obtained with additionof increasing amounts of biotin (Bohlscheid et al 2000) As suchother studies will be necessary to better understand the levels and

relative importance of key vitamins needed for good fermentation

0

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g C O 2 1 0 0 m L

Days

Mp+ALG804+Vit wo TP

Hv+ALG804+Vit wo TP

Mp+ALG804+Vit

Hv+ALG804+Vit

Fig 5 Effect of addition of YAN together with the complete vitamin mixture and with-out (wo) thiamine (T) and pantothenic acid (P) Fermentations were carried out at20 degC in the de1047297ned grape must results are the average and bars indicate the SD




performance Furthermore results which suggest that nitrogen andvitamins could play a role in fermentation kinetics of mixed culturesneed to be con1047297rmed with additional yeast strains

In summary the experimental model presented allows for modi1047297-cation of nutrient composition in a de1047297ned medium and then to mon-itor proportional changes in yeast populations using a simpledifferential plating medium based on WL nutrient agar It wasdemonstrated that in sequentially inoculated yeast fermentation a

NS strain inoculated prior to a S cerevisiae strain 1047297

rst acts to use nu-trients that eventually results in a sluggish or stuck fermentationYAN addition together with a vitamin mixture resulted in increasedS cerevisiae proportions in the mixed culture as well as an increasedfermentation rate These results could explain why the competitiveadvantage usually observed for S cerevisiae in mixed cultures waslimited by a nutrient availability caused by retention or removalfrom the medium by the NS strain Future work will be needed tobetter understand the role of vitamins and YAN levels (ammoniumand amino acids) in fermentation performance of mixed culturesand how strain variations affect the system

Acknowledgements

We would like to thank CSIC of the University of the Republic(UdelaR) Uruguay Group Project 656 for 1047297nancial support of thiswork and the ANII fellowship program for postgraduate students forsupporting Karina Medina

We are grateful for the critical review made for this manuscript byDr Paul Gill

References

Andorragrave I Berradre M Rozegraves N Mas A Guillamoacuten JM Esteve-Zarzoso B 2010Effect of pure and mixed cultures of the main wine yeast species on grape mustfermentations European Food Research and Technology 231 215ndash224

Anfang N Brajkovich M Goddard MR 2009 Co-fermentation with Pichia kluyveriincreases varietal thiol concentrations in Sauvignon Blanc Australian Journal of Grape and Wine Research 15 1ndash8

Backhus LE DeRisi J Brown P Bisson LF 2001 Functional genomic analysis of acommercial wine strain of Saccharomyces cerevisiae under differing nitrogenconditions FEMS Yeast Research 1 111ndash125

Barquet M Martiacuten V Medina K Peacuterez G Carrau F Gaggero C 2012 Tandemrepeat-tRNA (TRtRNA) PCR method for themolecular typing of non-Saccharomycessubspecies Applied Microbiology and Biotechnology 93 807ndash814

Barrajoacuten-Simancas N Giese E Areacutevalo-Villena M Uacutebeda J Briones A 2011 Aminoacid uptake by wild and commercial yeasts in single fermentations andco-fermentations Food Chemistry 127 441ndash446

Bataillon M Rico A Sablayrolles J-M Salmon J-M Barre P 1996 Early thiamin as-similation by yeasts under enological conditions impact on alcoholic fermentationkinetics Journal of Fermentation and Bioengineering 82 145ndash150

Bisson LF Kunkee R 1991 Microbial interactions during wine production In Zeikus JG Johnson EA (Eds) Mixed Cultures in Biotechnology McGraw-Hill New Yorkpp 37ndash68

Bohlscheid JC Edwards CG Fellman JK Ansen D 2000 Interactions betweenbiotin and nitrogen source on yeast growth and metabolism American Societyfor Enology and Viticulture Annual Meeting vol 51 AJEV Seattle WA p 301

Carrau F 2003 Characterization of yeast in relation to the ability to utilize nitrogenmdashstudies of aromacompounds PhD thesisin Chemistry Universidad de la Republica

Uruguay MontevideoCarrau F Neirotti E Gioia O 1993 Stuck wine fermentations effect of killersensitiveyeast interactions Journal of Fermentation and Bioengineering 76 67ndash69

Carrau F Medina K Boido E Farina L Gaggero C Dellacassa E Versini GHenschke PA 2005 De novo synthesis of monoterpenes by Saccharomycescerevisiae wine yeasts FEMS Microbiology Letters 243 107ndash115

Carrau F Medina K Farina L Boido E Henschke PA Dellacassa E 2008 Produc-tion of fermentation aroma compounds by Saccharomyces cerevisiae wine yeastseffects of yeast assimilable nitrogen on two model strains FEMS Yeast Research8 1196ndash1207

Ciani M Comitini F 2011 Non-Saccharomyces wine yeasts have a promising role inbiotechnological approaches to winemaking Annals of Microbiology 61 25ndash32

Ciani M Beco L Comitini F 2006 Fermentation behaviour and metabolic interac-tions of multistarter wine yeast fermentations International Journal of FoodMicrobiology 108 239ndash245

Ciani M Comitini F Mannazzu I Domizio P 2010 Controlled mixed culturefermentation a new perspective on the use of non-Saccharomyces yeasts inwinemaking FEMS Yeast Research 10 123ndash133

Clemente-Jimenez JM Mingorance-Cazorla L Martinez-Rodriguez S Las Heras-Vazquez FJ Rodriguez-Vico F 2005 In1047298uence of sequential yeast mixtures onwine fermentation International Journal of Food Microbiology 98 301ndash308

Diacuteaz-Montantildeo DM Favela-Torres E Coacuterdova J 2010 Improvement of growthfermentative ef 1047297ciency and ethanol tolerance of Kloeckera africana during thefermentation of agave tequilana juice by addition of yeast extract Journal of theScience of Food and Agriculture 90 321ndash328

Egli CM Edinger WD Mitrakul CM Henick-Kling T 1998 Dynamics of indigenousand inoculated yeast populations and their effect on the sensory character of Riesling and Chardonnay wines Journal of Applied Microbiology 85 779ndash789

Fleet GH 2003 Yeast interactions and wine 1047298avour International Journal of Food

Microbiology 86 11ndash

22Fleet GH 2008 Wine yeasts for the future FEMS Yeast Research 8 979ndash995Fleet GH Heard GM 1993 Yeasts-growth during fermentation In Fleet GH (Ed)

Wine Microbiology and Biotechnology Harwood Academic Publishers Switzerlandpp 27ndash54

Guzzon R Widmann G Settanni L Malacarne M Francesca N Larcher R 2011Evolution of yeast populations during different biodynamic winemaking processesSouth African Journal of Enology and Viticulture 32 242ndash250

Henick-Kling T Edinger W Daniel P Monk P 1998 Selective effects of sulfur diox-ide and yeast starter culture addition on indigenous yeast populations and sensorycharacteristics of wine Journal of Applied Microbiology 84 865ndash876

Henschke PA Jiranek V 1993 Yeast metabolism of nitrogen compounds In FleetGraham H (Ed) Wine Microbiology and Biotechnology Harwood AcademicPublishers pp 77ndash164

Jolly NP Augustyn OPH Pretorius IS 2003a The effect of non-Saccharomycesyeasts on fermentation and wine quality South African Journal of Enology andViticulture 24 55ndash62

Jolly NP Augustyn OPH Pretorius IS 2003b The use of Candida pulcherrima incombination with Saccharomyces cerevisiae for the production of Chenin blanc

wine South African Journal of Enology and Viticulture 24 63ndash69 Jolly NP Augustyn OPH Pretorius IS 2006 The role and use of non-Saccharomyces

yeasts in wine production South African Journal of Enology and Viticulture 2715ndash39

Jubany S Tomasco I Ponce De Leoacuten I Medina K Carrau F Arrambide N Naya HGaggeroC 2008 Towarda globaldatabase forthe molecular typing of Saccharomycescerevisiae strains FEMS Yeast Research 8 472ndash484

Lambrechts IG Pretorius IS 2000 Yeast and its importance to wine aromamdashareview South African Journal of Enology and Viticulture 21 97ndash129

Medina K Carrau FMGioia O Bracesco N 1997 Nitrogen availability of grapejuicelimits killeryeast growth and fermentation activity during mixed-culturefermentationwith sensitive commercial yeast strains Applied and Environmental Microbiology 632821ndash2825

Pallmann CL Brown JA Olineka TL Cocolin L Mills DA Bisson LF 2001 Use of WL medium to pro1047297le native 1047298ora fermentations American Journal of Enology andViticulture 52 198ndash203

Peacuterez G Farintildea L Barquet M Boido E Gaggero C Dellacassa E Carrau F 2011 Aquick screening method to identify β-glucosidase activity in native wine yeaststrains application of Esculin Glycerol Agar (EGA) medium World Journal of Microbiology and Biotechnology 27 47ndash55

Romano P Suzzi G Domizio P Fatichenti F 1997 Secondary products formationas a tool for discriminating non-Saccharomyces wine strains Antonie van Leeu-wenhoek International Journal of General and Molecular Microbiology 71239ndash242

Schutz M Gafner J 1993 Analysis of yeast diversity during spontaneous and inducedalcoholic fermentations Journal of Applied Bacteriology 75 551ndash558

Soden A Francis IL Oakey H Henschke PA 2000 Effects of co-fermentationwith Candida stellata and Saccharomyces cerevisiae on the aroma and composi-tion of Chardonnay wine Australian Journal of Grape and Wine Research 621ndash30

Swiegers JH Bartowsky PA Henschke PA Pretorius IS 2005 Yeast and bacterialmodulation of wine aroma and 1047298avour Australian Journal of Grape and WineResearch 11 139ndash173

Valle-Rodriacuteguez JO Hernaacutendez-Corteacutes G Coacuterdova J Estarroacuten-Espinosa M Diacuteaz-Montantildeo DM 2012 Fermentation of Agave tequilana juice by Kloeckera africanain1047298uence of amino-acid supplementations Antonie van Leeuwenhoek Interna-tional Journal of General and Molecular Microbiology 101 195ndash204

Viana F Belloch C Valleacutes S Manzanares P 2011 Monitoring a mixed starter of Hanseniaspora vineaendashSaccharomyces cerevisiae in natural must impact on2-phenylethyl acetate production International Journal of Food Microbiology151 235ndash240

Wang XD Bohlscheid JC Edwards CG 2003 Fermentative activity and productionof volatile compounds by Saccharomyces grown in synthetic grape juice mediade1047297cient in assimilable nitrogen andor pantothenic acid Journal of Applied Micro-biology 94 349ndash359

Zoecklein B Fugelsang K Gump B Nury F 1995 Wine Analysis and ProductionChapman amp Hall New York

Zohre DE Erten H 2002 The in1047298uence of Kloeckera apiculata and Candida pulcherrima yeasts on wine fermentation Process Biochemistry 38 319ndash324

Zott K Miot-Sertier C Claisse O Lonvaud-Funel A Masneuf-Pomarede I 2008Dynamics and diversity of non-Saccharomyces yeasts during the early stages inwinemaking International Journal of Food Microbiology 125 197ndash203

Zott K Thibon C Bely M Lonvaud-Funel A Dubourdieu D Masneuf-Pomarede I2011 The grape must non-Saccharomyces microbial community impact onvolatile thiol release International Journal of Food Microbiology 151 210ndash215




javanicaH occidentalis ( Jolly et al 2006) These yeasts generally havemoderate fermentative capacity and are important in the production of volatile aroma compounds (Viana et al 2011) There is concern aboutthe use of mixed cultures of NS apiculates and Saccharomyces in that theinitial rapid growth of some NS strains may have an inhibitory effect onsubsequent growth of S cerevisiae This phenomenoncould be implicatedin causing sluggish or stuck fermentations Nutrient limitation effects onmixed cultures wine fermentation have generally not been studied how-

ever some vitamins such asthiamine andpantothenic acid were associat-ed with sluggish wine fermentations (Bataillon et al 1996 Guzzon et al2011 Wang et al 2003) Therefore a very cautionary approach is cur-rently taken when considering use of NS apiculate strains in wineproduction

Metschnikowia pulcherrima can occur in high numbers in grapemust ( Jolly et al 2003b Peacuterez et al 2011 Schutz and Gafner1993) and are known to produce high concentrations of esters(Bisson and Kunkee 1991) especially the pear-associated esterethyl caprylate (Clemente-Jimenez et al 2005 Lambrechts andPretorius 2000) In addition production of undesirable volatilecompounds was not detected during mixed culture fermentationsusing M pulcherrima and S cerevisiae (Zohre and Erten 2002) anda positive sensory contribution to wine was shown for Chenin blancin sequential fermentations ( Jolly et al 2003b) However utilizingthe same treatment wines of Chardonnay were judged to be of an in-ferior quality compared with fermentation using only S cerevisiae

( Jolly et al 2003a)The aim of this study was to evaluate the effect of nutrient depletion

in a fermentation broth by NS strains during growth and fermentationin mixed cultures with S cerevisiae It is proposed that competition fornutrients is themain cause forinconsistentresults in NSndashSaccharomyces

co-fermentations We also suggest that understandingnitrogen balanceand vitamin requirements for the Saccharomyces inoculant could allowfor scaling-up the use of NS strains to the production level inwinemaking by helping to prevent sluggish or stuck fermentationsand improving the aroma composition and general positive attributesof the 1047297nal wine (Carrau 2003 Ciani et al 2010 Fleet 2008) Over500 strains of NS yeast were isolated from the Uruguayan winemaking

environment over a 5‐year period More than 20 of these NS strainsfrom grapesand theinitialstages of fermentation were previously iden-ti1047297ed that positively contributed to aroma and quality of white and redwines ( Jubany et al 2008 Peacuterez et al 2011) From this collection weselected two strains of the two main species present H vineae andM pulcherrima in order to study their potential application in simulta-neous or sequential fermentation with a conventional Saccharomyces

wine strain

2 Materials and methods

21 Yeast strains

The S cerevisiae strain used was ALG804 (DSM Denmark) a strainthat is used in commercial wine production NS strains used were H

vineae T025AF (Hv) isolated from wine fermentation of Tannat andM pulcherrima M0326 G (Mp) isolated from grape must of Merlotand both strains were previously characterized genetically (Barquetet al 2012)

Inocula were prepared in the same fermentation medium by incu-bation for 12 h on a rotary shaker at 150 rpm and 25 degC Inoculumsize was 5times105 cellsmL of medium except those indicated at 5times106

cellsmL Cultures were maintained at 4 degC on YEPD medium slants(1 yeast extract 2 peptone 2 glucose 2 agar containing 01 Mcitrate-phosphate buffer pH 45) Antagonistic or killer effect betweenstrains was analyzed using YEPD plating medium for killer neutral andsensitive determinations (Carrau et al 1993) resulting in a neutral

phenotype between these strains

22 Fermentation conditions

Yeast assimilable nitrogen (YAN) consumption pro1047297les for NSstrains were determined during fermentation of a Muscat of Alexan-dria white grape must containing 178 mg NL YAN 210 gL totalsugars and pH 35 The medium was 1047297lter sterilized using 045 μ m(Sartorius) and fermented in duplicate at 20 degC Static 125 mL batchgrape juice fermentations were done in 250 mL Erlenmeyer 1047298asks

(sealed with Muller valves 1047297

lled with pure sulfuric acid)The effects of nutrient availability during mixed culture fermenta-tions were studied using a previously described but modi1047297ed de1047297nedfermentation medium based on the nutrient composition of grape

juice (Carrau et al 2005) The modi1047297ed de1047297ned medium had abasic total nitrogen content of 50 mg NL with each amino acid andammonium component added in the same proportions previously in-dicated (Carrau et al 2005) The 1047297nal YAN concentration of 125 mg NL was made increasing the basic concentration by supple-mentation with diammonium phosphate (DAP) This YAN amountwas not a limiting concentration for complete fermentation of sugarsby strain ALG804 Equimolar concentrations of glucose and fructosewere added to 200 gL Addition of 300 mgL of DAP was done tofermentations in experiments where indicated in day three afterinoculation obtaining a 1047297nal YAN concentration of 188 mgNL inthese treatments Vitamin solutions were prepared and added to thefollowing 1047297nal concentrations (mgL) myo-inositol 100 pyridoxineHCl 2 nicotinic acid 2 calcium pantothenate 1 thiamine HCl 05PABA 02 Ribo1047298avine 02 biotin 0125 Folic acid 02 Thiamine andcalcium pantothenate were excluded from the vitamin solution whereindicatedErgosterol wasadded to a 1047297nal concentration of 10 mgL pre-viously solved in ethanol as described previously (Henschke and

Jiranek 1993) The 1047297nal pH of the medium was adjusted to 35 withHCl The medium was 1047297lter sterilized using 022 μ m (type Millipore)Static 125 mL batch fermentations were done in 250 mL Erlenmeyer1047298asks (sealed with Muller valves 1047297lled with pure sulfuric acid) at20 degC in duplicate to simulate wine making conditions Fermentationactivity was measured by CO2 weight loss expressed in grams per100 mL and decreasing YAN levels using the formaldehyde method

(Zoecklein et al 1995) Samples were taken once a day to measurecell growthin an improved Neubauer chamberRelative yeast cell num-bers distributions were determined during growth or fermentationusing the differential culture growth WL nutrient medium (Medinaet al 1997 Pallmann et al 2001) In this medium H vineae growthis identi1047297ed by its intense brilliant green color whereas M pulcherrima

acquires a light green and strain ALG804 a matt clear creamy color

23 Statistical analysis

ANOVA was done to establish signi1047297cance of differences for strainsinoculated in fermentation performance growth YAN consumptionand nutrient addition effects Statistica 71 software was used foranalysis

3 Results

31 YAN consumption in NS strains

YAN consumption pro1047297les for strains Hv Mp and ALG804 areshown in Fig 1A The YAN was consumed rapidly and completely inthree days by the Mp and ALG804 strains whereas YAN consumptionremained at ca 90 after this period for the Hv strain Fermentationand growth pro1047297les for the strains during YAN consumption is alsoshown in Fig 1B and C Although strain Hv had a higher fermentationrate than strain Mp YAN consumption was slower than that of strainMp The faster growth rate of strain Mp was consistent with its higherYAN consumption A reduced fermentation pro1047297le was seen for strain

Mp relative to that for strain Hv and both NS yeasts had only moderate
















0

05

1

15

2

0 1 2 3

g 1 0 0 m L C O

2

days

Fermentation

Mp

Hv

ALG 804

0

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0 1 2 3

x 1 0 6 c e l l s m L

days

Growth

Mp

HvALG 804

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YAN Consumption

Mp

Hv

ALG 804

mg NLA

B

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0 2 3 4 5 6 7 9 10

g

C O 2 1 0 0 m L

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0 2 3 4 5 6 7 8 9 10

g C O 2 1 0 0 m L

Days

ALG804Mp

Mp+ALG804

ALG804Hv

Hv+ALG804

A

B














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10

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0 1 3 6

S a c A L G 8 0 4

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75

95

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25

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0

10

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40

50

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80

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100

0 1 3 6

S a c A L G 8 0 4

Days


ALG804+Hv 5x10 5

ALG804+Hv 5x10 6

Hv 5x10 5 +ALG804

Hv 5x10 6 +ALG804


B

A


0

1

2

3

4

5

6

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0 2 3 4 5 6 7 9 10

g C

O 2 1 0 0 m L

Days

Mp+ALG804

Mp+ALG804+NH4

Mp+ALG804+NH4+Vit

0

1

2

3

4

5

6

0 2 3 4 5 6 7 8 9 10

g C O 2 1 0

0 m L

Days

Hv+ALG804

Hv+ALG804+NH4

Hv+ALG804+NH4+Vit

A

B


the SD





4 Discussion
















0

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0 1 2 3 4 5 6 7 8 10 11 12

g C O 2 1 0 0 m L

Days

Mp+ALG804+Vit wo TP

Hv+ALG804+Vit wo TP

Mp+ALG804+Vit

Hv+ALG804+Vit









Acknowledgements



References





























































0

05

1

15

2

0 1 2 3

g 1 0 0 m L C O

2

days

Fermentation

Mp

Hv

ALG 804

0

20

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120

140

0 1 2 3

x 1 0 6 c e l l s m L

days

Growth

Mp

HvALG 804

0

20

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80

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140

160

180

200

0 1 2 3days

YAN Consumption

Mp

Hv

ALG 804

mg NLA

B

C



0

1

2

3

4

5

6

0 2 3 4 5 6 7 9 10

g

C O 2 1 0 0 m L

Days

0

1

2

3

4

5

6

0 2 3 4 5 6 7 8 9 10

g C O 2 1 0 0 m L

Days

ALG804Mp

Mp+ALG804

ALG804Hv

Hv+ALG804

A

B














9930

40

47

15

25

40

80

0

10

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0 1 3 6

S a c A L G 8 0 4

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95

1012

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35

0

10

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0 1 3 6

S a c A L G 8 0 4

Days


ALG804+Hv 5x10 5

ALG804+Hv 5x10 6

Hv 5x10 5 +ALG804

Hv 5x10 6 +ALG804


B

A


0

1

2

3

4

5

6

7

0 2 3 4 5 6 7 9 10

g C

O 2 1 0 0 m L

Days

Mp+ALG804

Mp+ALG804+NH4

Mp+ALG804+NH4+Vit

0

1

2

3

4

5

6

0 2 3 4 5 6 7 8 9 10

g C O 2 1 0

0 m L

Days

Hv+ALG804

Hv+ALG804+NH4

Hv+ALG804+NH4+Vit

A

B


the SD





4 Discussion
















0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7 8 10 11 12

g C O 2 1 0 0 m L

Days

Mp+ALG804+Vit wo TP

Hv+ALG804+Vit wo TP

Mp+ALG804+Vit

Hv+ALG804+Vit









Acknowledgements



References


























































9930

40

47

15

25

40

80

0

10

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0 1 3 6

S a c A L G 8 0 4

Days


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75

95

1012

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35

0

10

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0 1 3 6

S a c A L G 8 0 4

Days


ALG804+Hv 5x10 5

ALG804+Hv 5x10 6

Hv 5x10 5 +ALG804

Hv 5x10 6 +ALG804


B

A


0

1

2

3

4

5

6

7

0 2 3 4 5 6 7 9 10

g C

O 2 1 0 0 m L

Days

Mp+ALG804

Mp+ALG804+NH4

Mp+ALG804+NH4+Vit

0

1

2

3

4

5

6

0 2 3 4 5 6 7 8 9 10

g C O 2 1 0

0 m L

Days

Hv+ALG804

Hv+ALG804+NH4

Hv+ALG804+NH4+Vit

A

B


the SD





4 Discussion
















0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7 8 10 11 12

g C O 2 1 0 0 m L

Days

Mp+ALG804+Vit wo TP

Hv+ALG804+Vit wo TP

Mp+ALG804+Vit

Hv+ALG804+Vit









Acknowledgements



References


















































4 Discussion
















0

1

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4

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6

7

0 1 2 3 4 5 6 7 8 10 11 12

g C O 2 1 0 0 m L

Days

Mp+ALG804+Vit wo TP

Hv+ALG804+Vit wo TP

Mp+ALG804+Vit

Hv+ALG804+Vit









Acknowledgements



References





















































Acknowledgements



References















































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