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MINI-REVIEW

What do we know about the yeast strains from the Brazilian

fuel ethanol industry?

Bianca Eli Della-Bianca & Thiago Olitta Basso &

Boris Ugarte Stambuk & Luiz Carlos Basso &

Andreas Karoly Gombert

Received: 16 October 2012 / Revised: 28 November 2012 / Accepted: 30 November 2012 / Published online: 28 December 2012# Springer-Verlag Berlin Heidelberg 2012

Abstract The production of fuel ethanol from sugarcane-based raw materials in Brazil is a successful example of a

large-scale bioprocess that delivers an advanced biofuel atcompetitive prices and low environmental impact. Two tothree fed-batch fermentations per day, with acid treatment ofthe yeast cream between consecutive cycles, during 68 months of uninterrupted production in a nonaseptic envi-ronment are some of the features that make the Brazilian

process quite peculiar. Along the past decades, some wildSaccharomyces cerevisiae strains were isolated, identified,characterized, and eventually, reintroduced into the process,enabling us to build up knowledge on these organisms. Thisinformation, combined with physiological studies in thelaboratory and, more recently, genome sequencing data,

has allowed us to start clarifying why and how these strainsbehave differently from the better known laboratory, wine,beer, and baker's strains. All these issues are covered in thisminireview, which also presents a brief discussion on future

directions in the field and on the perspectives of introducinggenetically modified strains in this industrial process.

Keywords Yeast. Fuel ethanol. Saccharomycescerevisiae. Industrial microbiology. Alcoholic fermentation

Introduction

Bioethanol and biodiesel are the main biofuels used world-wide today, with the first occupying an outstanding positionin the Brazilian economy. In the 1970s, as oil became moreexpensive and sugar cheaper, the Brazilian governmentestablished a national program (Pro-lcool), which aimed at

the substitution of gasoline with fuel ethanol (Goldemberg2008) produced by fermentation of sucrose from sugarcane.This initiative ensured Brazil about 30 years of expertiseahead of other countries in first-generation (1G) bioethanol

production and a privileged position in the fuel sector. Whilenowadays sugarcane 1G bioethanol replaces ~1 % of thegasoline used in the world, the potential of this technology isfar from being exhausted. Gains in productivity and geograph-ical expansion to larger areas may allow ethanol productionfrom sugarcane to replace ~10 % of the world's demand forgasoline before second-generation (2G) bioethanol production(from lignocellulosic biomass) reaches technological

maturity and possible economical competitiveness (Amorimet al. 2011; Buckeridge et al. 2012; Goldemberg 2007;Goldemberg and Guardabassi 2010; Soccol et al. 2010;Stambuk et al. 2008). Indeed, Brazilian sugarcane fuel ethanolis highly competitive when compared with production pro-cesses from other crops (e.g., corn and sugarbeet), as it showsthe highest percentage of greenhouse gas emission reduction,the highest energy balance and yields per hectare, and lower

production costs (Garoma et al.2012; Leal and Walter2010;Snchez and Cardona2008).

Bianca Eli Della-Bianca and Thiago Olitta Basso contributed equallyto this work

B. E. Della-Bianca :T. O. Basso : A. K. Gombert (*)Department of Chemical Engineering, University of So Paulo,PO Box 61548, 05424-970, So Paulo, SP, Brazile-mail: [email protected]

B. U. StambukDepartamento de Bioqumica, Universidade Federal de SantaCatarina, Florianpolis, SC, Brazil

L. C. BassoDepartment of Biological Sciences, University of So Paulo,Piracicaba, SP, Brazil

Present Address:

T. O. BassoNovozymes Latin America Ltda., Rua Professor FranciscoRibeiro, 683, CEP,83707-660, Araucria, PR, Brazil

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In the 2011/2012 crop, Brazil produced approximately23 billion liters of bioethanol and almost 60 % of all Brazilianlight vehicles can run on this fuel (Brazil 2012; BrazilianSugarcane Industry Association2012). The energy content ofsugarcane corresponds to 18 % of the country's energy matrixand represents a higher share than hydroelectricity (Altieri2012). This value includes the solid part, mainly bagasse,

which could be used as a substrate for 2G bioethanol produc-tion but is currently burned in boilers to generate electricity tothe mills and, if there is a surplus, to the national grid. Thissugar/energy market employs 4.3 million people (in both directand indirect jobs) and has a turnover of more than 80 billiondollars annually (Souza and Macedo 2010). Obviously, theachievement of such impressive numbers is only possible be-cause Brazil takes advantage of its soil characteristics, climaticconditions, and vast land area. Sugarcane crops are concentrat-ed in two regions: the Center-South, which produces approxi-mately 90 % of all sugar and ethanol, and the NorthNortheast,responsible for the other 10 % (Brazil2012).

Since 1975, the overall yield of the Brazilian fuel ethanolindustry has increased steadily by 34 % per year (Fig.1a),reaching nowadays over 6,500 lethanol per hectare of sug-arcane. This increase was possible due to several agriculturalimprovements, including selection of new sugarcane varie-ties with increased amounts of sugarcane biomass per hect-are (Fig. 1b). While the amount of total sugar per ton ofsugarcane also increased significantly during the first years(Fig. 1c), in the last 15 years, it seems to have reached a

plateau around 140 kg of sugar per ton of sugarcane. Thus,there is a need for improvement in sucrose production andaccumulation by the sugarcane plant, a feature that is under

current research in Brazil (Lam et al.2009; Waclawovsky etal. 2010). This trend in the amount of sugar per ton ofsugarcane biomass reflects directly into the amount of etha-nol produced from each ton of sugarcane, as the fermentationand distillation processes have also reached high industrialefficiency with an apparent plateau in the last years of ap-

proximately 80 l ethanol per ton of sugarcane (Fig.1d).During fermentation, the key step in bioethanol production,

sucrose (and glucose and fructose) is converted into ethanolby the yeast Saccharomyces cerevisiae in vats containingmillions of liters. When there are no major operational issues(such as rain, contamination, and power failure),yieldsas high

as 92 % of the stoichiometric conversion (0.511 gethanol/ghexose equivalent) can be achieved, yet the average industrialyield in Brazil has slightly decreased during the last decade(Fig.2). Since more than half of ethanol's final cost is due tothe cost of sugarcane, any increment on the already high yieldvalue would represent great economical gains. As an example,for an annual production of 30 billion liters of ethanol, an 1 %increase in fermentation yield would allow for the productionof extra 300 million liters from the same amount of rawmaterial or, in other words, from the same cultivated area.

Fig. 1 Evolution of the Brazilian fuel ethanol production industry. Thedata show the growth in overall yield (industrial+agricultural) (a), dueto improvements in the agricultural sector (b), including the selectionof higher sucrose-producing sugarcane varieties (c) as well as theindustrial fermentation performance (d) from 1975 to 2010 (source:Anurio estatstico da agroenergia. Ministry of Agriculture, Livestockand Supply. http://www.agricultura.gov.br/desenvolvimento-sustentavel/

agroenergia/estatistica Accessed 10 Jan 2012)

Fig. 2 Average ethanol yields (relative to 0.511 gethanolghexose1)from the Brazilian fuel ethanol production industry since the mid1970s. The 9092 % range is highlighted, with the aim of enabling abetter observation of the recent drops in the fermentation yield. Eachdata pointcorresponds to the average of values obtained from at least30 ethanol-producing plants, which were sampled every day (with aminimum of four samples per day) during at least 200 days in theseason (information kindly provided by Jaime Fingerut, Centro deTecnologia Canavieira, Piracicaba, SP, Brazil)

980 Appl Microbiol Biotechnol (2013) 97:979991
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One of the ways to achieve this increase is through theuse of adequate yeast strains, as has traditionally been donein wine and brewing industries. It has been proven thatstrains adapted to the harsh environment found in the indus-trial ethanolic fermentations are capable of delivering higheryields and productivities; studying these strains became anessential task for optimizing the process or having them

genetically modifiednot only for ethanol fermentationbut also for the production of other biofuels, chemicalprecursors, or higher value compounds from sugarcane-based substrates (e.g., artemisinin).

The peculiarities of the Brazilian fuel ethanol industrial

process

In Brazilian distilleries, sugarcane (Saccharum sp.) is the rawmaterial of choice for sugar and bioethanol production. It pro-vides readily fermentable sugars (1118 % wet w/w, with 90 %

of those being sucrose and 10 % glucose and fructose) that donot need any prior treatment to be metabolized byS. cerevisiae(Wheals et al. 1999). Moreover, endophytic nitrogen-fixing

bacteria can supply up to 60 % of sugarcane's nitrogen demandin low fertility soils, reducing the need for nitrogen fertilizersknown to require large amounts of fossil energy for their pro-duction. These features contribute to a highly favorable energy

balance (output:input ratio)sugarcane ethanol's energy bal-ance for Brazilian 2005/2006 crop season was estimated as9.3:1 and is predicted to be higher than 11:1 in 2020 (Macedoet al.2008), while for corn ethanol, it varies from less than 1:1(Pimentel2003) to 2.8:1 (Shapouri et al.2008).

Sugarcane juice and/or sugarcane molasses are used assubstrates for bioethanol production. After washing andchopping, sugarcane is crushed for juice extraction. Forsugar manufacturing, sucrose crystals are obtained afterintense juice evaporation and centrifugation. The remainingviscous phase is called molasses, which still contains 45 to60 % sucrose and 5 to 20 % glucose and fructose. Generally,the fermentation substrate is a mixture of varying propor-tions of sugarcane juice and molasses. This is because

molasses is nutrient-rich and can even provide a bufferingeffect during fermentation, while sugarcane juice can benutrient-deficient. On the other hand, molasses may alsocontain salts in high amounts and other inhibitors (mainly

browning reaction compounds) (Amorim et al.2009). Themineral composition of the substrate varies widely, depend-ing on molasses proportion, sugarcane variety and maturity,

soil, climate, and juice processing (Fig.3).Introduced in Brazil in the 1960s (Zanin et al.2000), the

Melle-Boinot process is the method of choice for ethanolfermentation and comprises three main features. First, fed-

batch mode is used due to higher production stability, sim-pler flow adjustment, and lower equipment costs whencompared to the continuous mode (Amorim et al. 2009;Drfler and Amorim2007; Godoy et al. 2008); however,according to Andrietta et al. (2007), the usual low-costadaptations of plants from batch to continuous modes mayhide the benefits from the latter option. Second, up to 9095 % of the yeast cells are recycled by centrifugation and

this allows high cell densities during fermentation (10 to17 % (wet w/v)), so that no intensive yeast propagation isneeded before (or during) each fermentation cycle. This,combined with the low nitrogen content of sugarcane-

based substrates, allows for an increase of only 5 to 10 %of yeast biomass during one fermentation cycle, which issufficient to replace the loss of cells during centrifugation.Accordingly, fermentation time becomes very short (6 to10 h) in comparison to other fermentation processes (e.g., 40to 50 h for corn ethanol), yet this benefit makes cooling thefermentors more difficult and temperatures go from 32 to35 C up to 40 C in the summer season (Amorim et al.

2004; Andrietta et al.2007; Laluce1991; Lima et al.1975,2001; Wheals et al. 1999). Lastly, recycled yeast goesthrough an acid treatment that decreases bacterial contami-nation (Amorim2005; Nepomuceno et al.1997). This treat-ment lasts 1 to 3 h and consists of dilution with water (1:1)and addition of sulfuric acid (pH 1.82.5), after which thecells are reused. This particular step occurs at least twice aday during a season that may last up to 250 days (Basso etal. 2008). In summary, fermentation starts by adding cane

Fig. 3 Mineral composition of sugarcane-based substrates.Black linesrepresent the concentration range found in the substrates, and gray barsshow the ideal concentration range for industrial production of ethanol.Data are given in mg/L of each element. *Ideal range is as low as

possible. **In form of NH4+ and R-NH2nitrogen. ***A toxicant; ideal

range for juice-only substrates. Data obtained from Amorim ( 2005)and Basso et al. (2011a)

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substrate, containing 18 to 22 % (w/w) total reducing sugars(TRS), to a 30 % (wet basis) yeast suspension that repre-sents 25 to 30 % of the fermentation vat total volume.Feeding time normally lasts for 4 to 6 h and fermentationis finished within 6 to 10 h, resulting in ethanol titers of 8 to12 % (v/v). Ethanol titers could be higher, but this wouldmean increasing fermentation time to unacceptable levels

and it would also compromise yeast viability and, conse-quently, its fitness during the subsequent fermentation cycle.

The fuel ethanol production process provides an environ-ment that is far from the optimal physiological condition foryeast. Several stress factors alternate during the process,among which the following are the most relevant ones(Fig.4): high sugar and ethanol concentrations, elevated tem-

peratures, pH variations, and presence of toxic compounds. Inthis unusual environment, yeast exhibits stress responsesagood industrial strain must be sufficiently robust to respondwell to these environmental variations, without altering itsfermentative characteristics over the cycles it is exposed to

during the whole crop season (Attfield1997). Sugarcane mustis an environment that does not preclude the proliferation ofcontaminants, mainly bacteria, given its high nutrient concen-tration, high water activity, and favorable pH for some species(Amorim2005). Besides bacteria, wild yeasts (including S.cerevisiaestrains) can contaminate and dominate the medium;after isolation and molecular and physiological characteriza-tions (Basso et al.2008), some of these strains were found to

be: (1) more adapted to the process than the starter cultures, (2)able to promote higher ethanol yields, and (3) not causative oftechnological issues, such as floculation and/or foaming.

Identity of Brazilian dominant and efficient fuel ethanol

yeast strains

One of the first attempts to determine which strains predom-inate in a group of five distilleries in Brazil was performed

by Basso et al. (1993). Using a karyotyping protocol adap-ted from Vezinhet et al. (1990), it was shown that starterstrains (baker's and other two S. cerevisiaestrainsTA and

NF) were unable to compete with indigenous yeasts thatcontaminated the industrial process. This study also showedthat a succession of different indigenousS. cerevisiaestrainswas detected throughout the fermentation season and that

only a wild strain (JA-1), previously isolated from onedistillery, could survive the recycle step. Similar observa-tions have been reported more recentlyda Silva-Filho etal. (2005b) concluded that only strains that were previouslyisolated from ethanol plants (and then used as starter cul-tures) were detected during almost the whole crop seasonand Basso et al. (2008) noticed that baker's yeast, tradition-ally used as a starter culture, was rapidly replaced by wildstrains in a short period (20 to 60 days) of cell recycle. Apartfrom karyotyping, other molecular techniques have also

been employed to investigate yeast population dynamicsduring ethanol fermentation, such as a PCR fingerprinting

method based on microsatellite (GTG)5 primer used by daSilva-Filho et al. (2005b), which was used to identify dom-inant indigenous strains.

Wild yeast contaminants have also been monitored in theethanol industry by molecular techniques during the fermen-tation step (Baslio et al.2008; Basso et al.2008; da Silva-Filho et al.2005b; de Souza Liberal et al. 2007) as well asduring the production of cachaa (a spirit made from sugar-cane juice) in several regions of Brazil (Badotti et al.2010;Bernardi et al.2008; Gomes et al.2007; Marini et al.2009).Da Silva-Filho et al. (2005b) reported on the presence of asignificant proportion of non-Saccharomyces contaminant

strains (1230 %) during a particular fermentation season.In distilleries in Northeastern Brazil, the most frequent con-taminants were identified asDekkera bruxellensis(de SouzaLiberal et al.2007; Leite et al.2012), responsible for severeimpacts on ethanol productivity. It is known that polyhex-amethyl biguanide (PHMB), a fungicide that affects the cellmembrane, killsD. bruxellensisstrains (Elsztein et al.2008)

but not the industrial fuel ethanol strain JP1 (although strainPE-2 is sensitive to this compound). Strain JP1 tolerance toPHMB was later found to be related to the induction ofgenes involved in cell wall integrity and in general andoxidative stress responses (Elsztein et al.2011).

In the same region, Candida tropicalisand Pichia galei-formis were also detected as major yeast contaminants in acutecontamination episodes, being responsible for decreased eth-anol yields (Baslio et al. 2008). However, it should bestressed that in a wider study covering Central-SoutheasternBrazil, in only a few distilleries could non-Saccharomycescontaminants be identified (mainly Schizosaccharomyces

pombe,D. bruxelensis, andC. krusei), representing less than5 % of the yeast population (Basso et al. 2008). It wasdemonstrated that such contaminant non-Saccharomyces

Fig. 4 Environmental stresses found by yeast during alcoholic fer-mentation phases in the Brazilian fuel ethanol production process; Ahigh sugar concentration, B high ethanol concentration, C osmoticstress and bacterial contamination, D high temperature, E acid stress(adapted from Gibson et al. (2007))



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strains were more frequently found in distilleries operatingwith relatively lower final ethanol concentrations (6 % v/v)and would not pose a problem for regularly operating ethanol

plants that work with higher ethanol titers.It is generally accepted by researchers and industrial

personnel involved in large-scale ethanol production thatstarting the fermentation season with adapted (selected)

strain(s) is of crucial importance in order to keep yeastpopulation stability, which, in turn, leads to reproducible(constant) ethanol yield and productivity. Thus, the largeinoculum required at the beginning of the season (starter) iscommonly prepared by mixing 2 to 12 t of baker's yeast with10 to 300 kg of selected strains in active dry yeast form. Oneexample is the study performed by da Silva-Filho et al.(2005a), which describes the use of both PCR fingerprintingand physiological assays in the selection of an indigenousstrain (JP1) as a proposed starter culture. This strain showedstress tolerance (towards acidic pH, ethanol, and high tem-

perature conditions) and fermentative capacity similar to

those of other commercial industrial strains. After reintro-duction as the starter strain in the distillery it had beenisolated from, it was found that JP1 strain was able todominate the population while conferring high ethanolyields (>90 %) for two consecutive production seasons.

Although these results might suggest that selecting well-fitted indigenous strains is an attractive strategy to guaranteehigh product yields and population homogeneity duringindustrial fermentations, one should be aware that such atask is quite laborious and not always successful. First, theselected strain must have appropriate physiological charac-teristics, such as good fermentation performance, high dom-

inance, and high persistence. In this context, dominance isthe proportion of a particular strain inside the fermentor at agiven point of the season and is related to competitivenessamong strains and to their specific growth rate. In contrast,

persistence means the strain's ability to withstand the multi-ple stress conditions found in the bioethanol fermentationprocess and, therefore, survive throughout the season andcan be expressed as the proportion of distilleries in which a

particular strain was able to be implanted, or implantationcapability. Second, the strain must possess appropriate tech-nological properties, like absence of flocculation and ofstuck fermentations, and low foam formation. In the ethanol

production process adopted in Brazil, flocculating yeast canincrease fermentation time because they cannot reach thesubstrate as efficiently as nonflocculating cells as well ascause clogging and lower centrifugation yield. AlthoughPereira and Cunha (2000) and Gomes et al. (2012) con-structed flocculating ethanol strains, suggesting the use ofan approach more similar to the repitching performed by the

brewing industry and a decrease in cell recycle costs, thiswould require equipment adaptation in current Brazilianethanol plants.

The 12-year yeast selection study performed by Basso etal. (2008) also made use of karyotyping as a tool to spot outdominant indigenous strains at various ethanol producing

plants. Surprisingly, among more than 300 dominant iso-lates, only 20 % showed appropriate technological charac-teristics and were further evaluated at laboratory conditionsthat simulated the industrial process. Of these strains, only

14 presented good physiological properties. These strainswere then reintroduced in the distilleries they had beenisolated from and also in other ethanol plants in order toevaluate their implantation capability throughout successive

production seasons. This program selected, among others,the strains PE-2 and CAT-1, which showed the highestdominance during the research time frame. These twostrains currently represent 70 % of the Brazilian market forfuel ethanol yeast strains (Henrique Amorim, personal com-munication). Besides these, four other strains (SA-1, BG-1,FT858, and JP1) are commercially available (LNF-LatinoAmericana Ltda. and Marcos Morais Jr., personal commu-

nication). According to Basso et al. (2008), it was not possibleto establish a relationship between strain adaptability and

particular process features, such as mode of operation andtype of substrate. On the other hand, da Silva-Filho et al.(2005a) showed a relationship between the prevalence of a

particular strain and the type of substrate used in the industrialplant, in this case, juice- or molasses-based medium.

Physiology of Brazilian industrial fuel ethanol strains

Until the mid 1990s, IZ-1904, TA and baker's yeast were the

most used starter strains for industrial fuel ethanol fermen-tation. However, in laboratory studies, their physiological

parameters were obtained after a single fermentation round(without cell recycle). When the same strains were evaluatedunder cell recycle conditions, valuable physiological dataarose. Indeed, when compared with baker's yeast, IZ-1904

presented higher ethanol yield (and titer) and also lowerbiomass gain, but higher glycerol formation, lower viability,and very low levels of intracellular trehalose. Such datasuggested that IZ-1904 would not be able to endure cellrecycle, since the higher ethanol yield observed was at theexpense of biomass formation and trehalose accumulation

(Alves1994; Basso et al.1988). These data were the startingpoint for studies on the implantation capability of thesestarter strains in distilleries, and further results demonstratedthat the only strain able to persist in industrial fermentationconditions was JA-1, a strain previously isolated from anethanol plant (Basso et al.1993). When compared with TA,JA-1 showed superior fermentation capabilities, such ashigher ethanol yield, lower glycerol formation, higher cellviability, and higher trehalose content (Basso and Amorim1994).



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PE-2 and VR-1 strains were first introduced in the 1995/1996 season in 24 distilleries, showing a remarkable implan-tation capability. These strains represented 80 to 100 % of thetotal yeast biomass in the fermentors of 12 distilleries andwere implanted in 63 % of the plants, accounting for 42 % ofthe biomass at the end of the season (Basso et al.1996). Sincethen, PE-2 strain has been used as a reference industrial strain

and, therefore, compared with baker's yeast to disclose phys-iological traits that could be related to its superior fermentation

performance. It also proved to be suitable for microvinifica-tion of raspberry juice (Duarte et al.2010) and sweet potatohydrolysate (Pavlak et al. 2011). Its high ethanol tolerancemade PE-2 appropriate for very high gravity (VHG) fermen-tation, attaining final ethanol contents of >19 % (v/v) with a

productivity of >2.5 gl1h1, while the laboratory CEN.PK113-7D strain reached 17.5 % (v/v) ethanol with a productiv-ity of 1.7 gl1h1 (Pereira et al.2010b,2011). This strain wasalso studied under VHG fermentations with cell recycle,attaining high ethanol titer and productivity during 15 cycles

(Pereira et al.2012). Also, a recombinant derivative of PE-2bearing theFLO1gene, responsible for a flocculation protein,performed successfully under VHG and flocculationsedi-mentation cell recycle conditions (Gomes et al.2012).

These industrial strains usually accumulate high levels ofstorage carbohydrates (trehalose and glycogen, which canaccount for 20 % w/w of yeast dry weight) towards the endof fermentation, as demonstrated by Paulillo et al. (2003).The degradation kinetics of these storage carbohydrates instrains SA-1 and PE-2 was analyzed and, under high tem-

perature and high biomass concentration, these compoundscould be further metabolized. This, in turn, resulted in extra

ethanol formation and a consequent increase in proteinlevels, which went from 35 to 42 % (w/w) and from 42 to52 % (w/w) in PE-2 and SA-1 strains, respectively. Thisstrategy is outlined as an important way to add economicalvalue to the excess yeast slurry commercialized by theethanol industry as animal feed, in which the protein levelsare expected to be at least 40 % (Amorim and Basso1991).

Regarding the patterns of sugar utilization by industrialfuel ethanol yeasts, their ability to ferment sucrose (Mirandaet al.2009) and maltose and maltotriose (Amorim Neto et al.2009; Duval et al. 2010) has been evaluated. The resultsindicated that strain CAT-1 ferments maltose but not malto-

triose, and it showed similar fermentation performance andhigher thermotolerance when compared to standard com-mercial whisky-distilling yeast (Amorim Neto et al. 2009).These industrial yeast strains can also easily ferment sugar(glucose) in high concentrations, up to 330 g/l for strain PE-2 (Pereira et al.2010a).

Alves (2000) highlighted the high rate of sucrose hydro-lysis by baker's yeast in relation to PE-2 strain duringfermentation, in both cases exceeding glucose and fructoseuptake capacity. Accordingly, a greater accumulation of

hexoses (nearly double) was observed for baker's strain inrelation to PE-2 strain. It was proposed that this conditionwould be imposing a higher osmotic stress and thus, ac-counting for the higher glycerol production observed incultivations with baker's yeast; internal glycerol was alsomuch higher during fermentation with this strain. It was alsodemonstrated that both strains were deprived of a succinic

acid transporter. Nevertheless, when they were placed in14C-succinate-containing medium, intracellular radioactivitywas twice higher in baker's yeast, suggesting a less selectivemembrane than PE-2's. During cell recycle, PE-2 presentedhigher performance than baker's yeast, as indicated by phys-iological and technological parameters (Basso et al. 2008)such as higher ethanol yield, lower glycerol formation,vigorous growth, and high cell viability, presumably pro-moted by its higher levels of intracellular trehalose andglycogen. Compared to the laboratory strain CEN.PK113-7D, the PE-2 strain could indeed accumulate higher contentsnot only of trehalose and glycogen (1.1- and 2.6-fold, re-

spectively) but also of sterols (threefold), which may beanother form of adaptation to the stressful VHG conditionsthat the strains have been exposed to (Pereira et al. 2011).

PE-2 strain indeed showed higher viability and trehaloselevels in cell recycle conditions when compared to anotherBrazilian industrial strain, M26, but few differences regard-ing ethanol yield (Dorta et al. 2006). On the other hand,M26 presented higher production of succinic acid, what can,at least, in part, explain the higher antibacterial effect pre-viously observed against Lactobacillus fermentum CCT1396 (de Oliva-Neto et al. 2004) and L. fermentum CCT1407 (Cherubin2003). Dorta et al. (2006) also showed that

low pH (3.6) and high ethanol titers (9.5 % w/v), actingsynergistically, were the major stress factors to decreaseindustrial yeast cell viability and that low pH (3.6) was themain factor to decrease ethanol yield.

Apart from presenting the population dynamics thatresulted in the isolation of strain JP1 (described in the

previous section), da Silva-Filho et al. (2005a) also studiedseveral of its physiological parameters. They showed thatunder laboratory conditions, JP1 displayed ethanol yieldssimilar to those observed for industrial fuel strains BG-1,CR-1, PE-2, and SA-1 (ranging from 0.46 to 0.50 gethanol/gsucrose). It was also verified that JP1's tolerance towards

acid, heat, and ethanol stresses are comparable to PE-2'sresponse and that a temperature increase from 37 to 42 Cmagnified cell viability decrease caused by low pH or highethanol concentration. In a molasses-based medium, JP1grew slower than PE-2 (0.16 vs. 0.23 h1), whereas theyexhibited similar growth rates on diluted cane juice medium(around 0.3 h1), these observations probably reflect thedistinct environments these strains were isolated from.

The majority of the (few) papers published on the phys-iology of industrial strains employed in Brazil investigated



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the response of these strains towards stress conditions asso-ciated with the industrial scenario, such as high ethanoltiters, elevated temperature, high osmotic stress due to sugarand salts, low pH, possible inhibitors, and bacterial contam-ination. These conditions have been investigated in moredetail for other fuel ethanol-related strains, such as the onesinvolved in corn-based ethanol industry (Zhao and Bai

2009). For baker's strain, temperature, pH, sugar concentra-tion, nitrogen (Alves1994; Gutierrez1989,1991), potassi-um, sulfite (Alves1994), nitrite (Gutierrez and Orelli1991),2,4-dinitrophenol (Gutierrez 1989, 1991), benzoic acid(Gutierrez et al. 1991a), acetic acid (Gutierrez et al.1991b), and octanoic acid (Gutierrez1993) had their effectsinvestigated. Noteworthy, sulfite up to 300 mg/l slightlyincreased glycerol formation but did not affect ethanol yield,yeast viability, or cellular trehalose levels, possibly becauseit reduced bacterial growth and, consequently, lactic andacetic acid concentrations in the culture medium (Alves1994).

Some metals, such as aluminum and cadmium, can alsoaffect industrial strains. The toxic form of aluminum (Al3+)is favored by the acidic environment of fermentation and,although very low levels of cadmium were detected insugarcane from crops fertilized with municipal sewagesludge, toxic levels of this element were found during fer-mentations, since yeast has the ability to accumulate cadmi-um throughout the intensive cell recycle. Among the toxiceffects of both metals are lower cell viability, decreasedintracellular trehalose content, and reduced ethanol yield.While the presence of magnesium only minimizes alumi-num toxicity, the use of a molasses-rich medium eradicates

it. The use of vinasse/stillage (effluent from ethanol distil-lation) is efficient to alleviate both aluminum and cadmiumtoxic effects. When compared to baker's yeast, strain PE-2

presents higher tolerance and lower intracellular accumula-tion of aluminum during cell recycle; concerning cadmiumtoxicity, it is more tolerant than strain IZ-1904 (Basso et al.2004; de Souza Oliveira et al. 2009; Mariano-da-Silva andBasso2004; Silva et al. 2010).

Excessive yeast growth observed in some distilleries com-promised ethanol yield; for this reason, increasing ethanolyield by loweringbiomass and/or by-product formation (main-ly glycerol) was performed by addition of 2,4-dinitrophenol

(Gutierrez1989), acetic acid (Gutierrez et al.1991b), or ben-zoic acid (Gutierrez et al. 1991a). The latter treatment wasmore efficient and kept high yeast cell viability. During the1992/1994 crop seasons, it was used in three different distill-eries and evaluated in laboratory scale, simulating cell recycle

process and using baker's yeast (Basso et al. 1996). In alldistilleries, a significant increase in ethanol yield and a reduc-tion in glycerol formation were observed during the initialcycles, but later on, bacterial growth was high enough to makethe use of benzoic acid in ethanol plants impracticable. In

laboratory conditions, not only the same trend was observedbut also a drastic reduction of succinic acid formation by yeastwas noticed. Additionally, it was demonstrated that succinicacid produced by yeast exerts an antibacterial effect duringfermentation, being an important inhibitor of lactic acid bacte-ria (Basso et al.1997). An ecological reason for succinic acid

production was then suggested, since it renders yeast more

competitive in an industrial fermentation environment, espe-cially in fuel ethanol distilleries where bacterial contaminationis very frequent.

Bacterial contamination is often regarded as a majordrawback during industrial ethanol fermentation. Besidesdeviating feedstock sugars from ethanol formation, thereare also detrimental effects of bacterial metabolites (suchas lactic and acetic acids) upon yeast fermentative perfor-mance, resulting in reduced ethanol yields, yeast cell floc-culation, and low yeast viability. Most of the bacterialcontaminants of the fermentative step of ethanol productionare lactic acid bacteria, probably because they are more able

to cope with low pH values and high ethanol concentrationswhen compared to other microorganisms; indeed,

Lactobacillus was the most abundant genera isolated fromBrazilian ethanol plants, from both homo- and heterofer-mentative types (Basso et al. 2011a).

First genomic information of Brazilian fuel ethanol yeast

strains

Industrial strains are a rich source of unexplored geneticdiversity, mainly for their alleles and/or mutations control-

ling traits relevant to the industrial fermentation. Identifyingand characterizing these mutations could provide the basisfor a reverse metabolic engineering (ME) approach in orderto develop even better fermenting strains. The detailed ge-nomic structure of some of these industrial strains hasstarted to be determined (Argueso et al. 2009; Stambuk etal.2009), revealing significant structural and sequence var-iations when compared to laboratory strains or others iso-lated from nature. For example, while many brewing andwine industrial yeast strains are interspecies hybrids be-tween two or even three yeast species (S. cerevisiae, S.bayanus, and/orS. kudriavzezii), generating polyploid, an-

euploid, or even allopolyploid genomes with sometimesmosaic chromosomes (Borneman et al. 2011; Novo et al.2009; Querol and Bond2009), the industrial fuel ethanolyeasts selected in Brazil are bona fide heterothallic diploidS. cerevisiaestrains, exceptions being strains JP1, which is ahomothallic diploid (Reis et al. 2012), and FT-858, a new

polyploid strain probably derived from PE-2 that exhibited ahigh implantation rate during the 2011/2012 crop season(Henrique Amorim, personal communication). The genomefrom strain BG-1 contains two S. paradoxus introgression



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events; however, this strain is not an interspecific hybrid.These introgression events encompass regions containing

MAL(maltose utilizing) genes andSTL1(glycerol transport)and PAD1 and FDC1 (both phenolic acid decarboxylases)genes, all of them relevant to the industrial conditions (Dunnet al.2012). A list of all Brazilian industrial strains whichgenomic data have already been published is shown in

Table1.The complete genome sequence of a haploid derivative of

strain PE-2 (JAY291) is published (Argueso et al. 2009),and the diploid PE-2 strain is already sequenced (GonaloA. Pereira, personal communication). More recently, thecomplete genome of the diploid strain CAT-1 was alsodetermined (Babrzadeh et al.2012). The genomes of thesefuel ethanol strains were found to be highly heterozygous(around 23 SNPs/kb), affecting over half of the predicted

protein-coding genes present in the S. cerevisiae referencegenome (from laboratory strain S288c). These industrialyeast strains also display significantly less transposable

elements in their genomes, several structural polymor-phisms between homologous chromosomes, and largeregions of loss of heterozygosity, when compared to theS288c strain (Argueso et al. 2009; Babrzadeh et al.2012;Borneman et al. 2011). In fact, chromosomal rearrange-ments were identified earlier in PE-2 strain (Lopes et al.2002) and in JP1 strain (Lucena et al. 2007), both in the

industrial environment and when cultivated for long periodsin laboratory conditions. It was observed, however, thatsince these chromosomal rearrangements occur near chro-mosome ends, i.e., in regions that do not contain essentialgenes, they are unlikely to impair meiosis (Argueso et al.2009); instead, they contain amplifications that improvefitness for industrial fermentations (Argueso et al. 2009;Stambuk et al.2009). It was proposed that these polymor-

phisms could play a role in the higher adaptability of thesevariants throughout the fermentation process (Basso et al.2008), contributing to their higher productivity. Althoughnot observed by Lopes et al. (2002), this was further

Table 1 Brazilian fuel ethanol strains and their genomic details

Strain Ploidy Detailsa References Databasesb

PE-2 Diploid Heterothallic; presents efficient sporulation and ~2.6 SNPs/kb

between allelic regions in homologous chromosomes

Argueso et al. (2009) GEO accession nos. GSE14601

and GSE17578

Carries amplified SAM3/SAM4 and SNO/SNZ Stambuk et al. (2009) GEO accession no. GSE13875

JAY291 Haploid Presents 5.4 SNPs/kb in comparison to S288c Argueso et al. (2009) GenBank accession no.

ACFL01000000ho FLO8 HAP1 SUC2 Babrzadeh et al. (2012)

Carries amplified SEO1and ADH7

Does not carry RTM1

CAT-1 Diploid Heterothallic; presents high heterozygozity and large-scale lossof heterozygozity but no sequences corresponding to the

2-m plasmid

Stambuk et al. (2009) GEO accession no. GSE13875

Carries amplified SEO1, ADH7, RDS1, SGE1, ARR1-ARR3,

SAM3/SAM4, SNO/SNZ

Babrzadeh et al. (2012) SRA accession no. SRA012578

Carries lower copy number ofMPH2, SOR2, HXT15, MPH3,

SOR1, HXT16, DAK2, AGP3, SNO4, ERR3, AAD15, BDS1,

FIT2, FIT3,FRE5, PHR1, MAL11(AGT1),HXT6,HXT7,

ENA1, ENA2, ENA5, CUP1-1, CUP1-2

Does not carry RTM1, FLO1orFLO9

Missing regions in FLO5, FLO10and FLO11

BG-1 Diploid Heterothallic; carries a smaller version of the 2-m plasmid Stambuk et al. (2009) GEO accession no. GSE13875

AmplifiedSNO/SNZ Dunn et al. (2012) GEO accession no. GSE26689

PresentsS. paradoxus introgression regions containing MAL,

STL1, PDA1and FDC1

SRA accession no. SRA049752

Missing regions in mitochondrial COX1,ENA and HXT6/7

JP1 Diploid Homothallic Da Silva-Filho et al. (2005a)

Reis et al. (2012)

VR-1 Diploid AmplifiedSNO/SNZ Stambuk et al. (2009) GEO accession no. GSE13875

Swinnen et al. (2012) SRA accession no. SRA049724

SA-1 Diploid Amplified SNO/SNZ Stambuk et al. (2009) GEO accession no. GSE13875

aGene amplifications, altered copy number, and missing regions are described in comparison to S288c genome sequenceb Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/), GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and Sequence ReadArchive (SRA;http://www.ncbi.nlm.nih.gov/sra)

http://www.ncbi.nlm.nih.gov/geo/http://www.ncbi.nlm.nih.gov/genbank/http://www.ncbi.nlm.nih.gov/srahttp://www.ncbi.nlm.nih.gov/srahttp://www.ncbi.nlm.nih.gov/genbank/http://www.ncbi.nlm.nih.gov/geo/


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investigated by Argueso et al. (2009), who showed that suchcharacteristics contributed to high ethanol and cell mass

production as well as to high temperature and oxidativestress tolerance, probably accounting for their high adapta-tion to the industrial environment.

Another important feature is the amplification of telo-meric SNO and SNZgenes, shared by five industrial strains

(PE-2, CAT-1, BG-1, SA-1, and VR-1) (Stambuk et al.2009). These genes are involved in thiamine (vitamin B1)and pyridoxine (vitamin B6) biosyntheses, required for sug-ar catabolism by yeast. An increased copy number of thesegenes contributed for efficient growth under thiamine re-

pression, when in medium lacking pyridoxine and with highsugar concentration. Surprisingly, these five strains did notshow amplification of the SUC2gene (Stambuk et al.2009),as seen for telomeric SUC genes in baker's and distillers'strains adapted to sucrose-rich broths (Bentez et al. 1996;Codn et al.1998). As SUC2 encodes for an extracellularinvertase, this indicates that sucrose conversion is not a limit-

ing step for these strains during industrial fermentation.RTM1, another gene usually found in association with telo-mericSUCgenes and implicated in Resistance ToMolassesof baker's and distillers' yeasts by an unknown mechanism(Denayrolles et al.1997; Ness and Aigle1995), is also absentfrom the JAY291 genome (Argueso et al.2009; Borneman etal. 2011). In the BG-1 strain genome, there are presumeddeletions in regions containing ENAgenes (as corroborated

by unpublished results from our laboratory, which show alower osmotic tolerance of this strain) and in the HXT6/7locus (Dunn et al.2012).

Swinnen et al. (2012) applied pooled segregant whole-

genome sequence analysis to map all the quantitative traitloci (QTL) that determine the high ethanol tolerance (up to17 % ethanol) presented by a segregant of strain VR-1. Themain genes associated with this trait were MKT1, which hasa regulatory role in global gene expression, and APJ1,which encodes for an Hsp40 family protein with a negativeeffect on ethanol tolerance.

Conclusions and perspectives

The Brazilian ethanol production process is quite peculiar,

given its features like yeast cell recycle and acid treatment.Analysis of strains isolated from this process shows us thatsuccessfully implementing a modified laboratory strain foruse in this industry can be very difficult, if not impossible,since even some highly dominant isolates, as PE-2, are nowdisappearing from the distilleries and giving space to more-adapted ones.

In this sense, applying evolutionary engineering techniquesto these industrial strains can be challenging, as they have beenevolving since decades in the process itself. Probably just as

challenging is the implementation of metabolically engineeredstrains. ME could be used not only to increase their ethanol

productivity and/or yield but also to change substrate utilizationor the desired end product while keeping their highly desiredtolerance traits. Examples of ME targets would be heterologousenzyme expression for production of 2G ethanol, biopolymers(e.g., polyhydroxyalkanoates (PHA)and polyhydroxybutyrates

(PHB)) or other biofuels (e.g., synthetic diesel and butanol),consequently upgrading sugar/ethanol distilleries to biorefi-neries. However, one should consider the regulatory issuesinvolved in the industrial use of genetically modified (GM)strainsrecently, one process for farnesene production fromsugarcane-based media by GM yeast was approved by theBrazilian biosafety commission (Brazil2010).

Presently, there are many aspects about the Brazilianethanol strains that remain unknown, such as why theydominate the fermentors so quickly in some distilleries,

but in 40 % of the plants, none of them can be implanted.At the same time, these questions should be the object of

more research, and there is the need of isolating or produc-ing new strains, since the number of commercially availablestrains for this industry is rather small. One alternative to thechallenge of finding a strain versatile enough to beimplanted in multiple distilleries is the isolation of custom-ized strains in an in-house manner, selecting the ones thatare already at the plants and probably more fitted to thedistinct environment they encounter every day. It is impor-tant to keep in mind that, among the more than 400 millscurrently in operation in Brazil, there are no two identicalindustrial processes.

Isolating new suitable strains is also challenging because

the sugar and ethanol production processes are constantlychanging, mainly due to: new sugarcane crops, different

proportions of sugarcane juice to molasses used in substrateformulation, different molasses composition (due to im-

proved sucrose extraction during edible sugar production),and increased mechanical harvesting that might bring other

plant materials to the crushing step, such as tops and leaves,among others.

The possibility of using GM strains for industrial fuelethanol production is highly dependent on the perspectivesof implementing assepsis in the different stages of the pro-cess, since wild yeasts with higher specific growth rate and

viability will most probably outcompete the GM strains.Currently, the price of ethanol does not justify such practi-ces, but what would happen if we had GM strains withimproved performance in a scenario of high gasoline prices?A laboratory strain was already engineered and evolved foran 11 % higher ethanol yield on sucrose (Basso et al.2011b), and this strategy was recently implanted in anindustrial strain and is currently being tested under industrialconditions, with promising results (Boris Stambuk's unpub-lished data).



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To conclude, we can affirm that we are still at the begin-ning of understanding what makes these industrial strainsdominate and persist (or not) inside the industrial vats. Moregenome sequencing and annotation, targeted physiologicalstudies, and also field trials are required to fill this gap. Last

but not least, we do not even know where these wild yeaststrains come from: do they have an origin in the sugarcane

fields, are they brought by insects that visit the productionplant or, since they are more related to baker's strains than towine or brewing yeast (Stambuk et al. 2009), did theysimply evolve from commercial baker's yeast?

Acknowledgments We would like to thank Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP, Brazil) for grants withinthe BIOEN framework and Conselho Nacional de DesenvolvimentoCientfico e Tecnolgico (CNPq, Brazil) and Coordenao de Aperfei-oamento de Pessoal de Nvel Superior (CAPES, Brazil) for the schol-arships provided.

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