Using the residue of spirit production and bio-ethanol for protein production by yeasts

Waste Management 31 (2011) 108–114

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Using the residue of spirit production and bio-ethanol for protein production by yeasts Cristina F. Silva a,⇑, Silvio L. Arcuri a, Cássia R. Campos a, Danielle M. Vilela a, José G.L.F. Alves b, Rosane F. Schwan a a b

Departamento de Biologia, Universidade Federal de Lavras, 37200-000 Lavras, MG, Brazil Departamento de Ciência dos Alimentos, Universidade Federal de Lavras, 37200-000 Lavras, MG, Brazil

a r t i c l e

i n f o

Article history: Received 28 February 2010 Accepted 12 August 2010 Available online 22 September 2010

a b s t r a c t The residue (vinasse) formed during the distillation of bio-ethanol and cachaça, a traditional rum-type spirit produced from sugar-cane in Brazil, is highly harmful if discharged into the environment due to high values of COD and BOD. One possibility for minimizing the impact of vinasse in soils and waters is to use the residue in the production of microbial biomass for use as an animal feed supplement that will provide high levels on nitrogen (>9% d.m.) and low content of nucleic (610% d.m.) This paper reports the production and quality of biomass produced from fermentation of Saccharomyces cerevisiae and Candida parapsilosis in culture media under 12 different culture conditions and the respective effects of each variable (glucose, yeast extract, peptone, potassium phosphate, vinasse, pH and temperature). Of the S. cerevisiae isolates tested, two (VR1 and PE2) originating from fuel alcohol-producing plants were identified as offering the best potential for the industrial production of single cell protein from vinasse due to highest biomass productivity. Our results showed a potential viable and economic use of vinasse. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Brazil is the largest producer of sugar-cane and its products, followed by India, China, Thailand, Mexico, Kenya and Pakistan (Boletim de Economia e Politica Internacional, 2010). These countries and over 200 other countries grown sugar-cane to produce sugar, alcohol and/or bio-ethanol. In Brazil there is increasing demand from small-scale production of a typical drink known as cachaça, a rum-type spirit, in addition to bio-ethanol. Brazil is a huge producer of clean energy due to reductions of CO2 emissions and is increasing the implementation of bio-ethanol plants in various countries in Latin America and Africa (Anuário estatístico da Agroenergia, 2009). The production of cachaça and bio-ethanol generates a residue known as vinasse or stillage in the ratio of 4–10 L of residual L 1 of product (Shojaosadati et al., 1999). It is estimated that in 2010 there will be a production of 85.9 billion gallons of ethanol, representing an increase of 16% (www.sugarcaneblog.com/2010/) generating about 687.2 billion liters of vinasse. Although vinasse is classified as a non-inert, biodegradable residue (class IIA; ABNT, 2004), its chemical oxygen demand is elevated by high concentrations of soluble material and non-volatile fermentation by-products that are extremely recalcitrant in nature ⇑ Corresponding author. Tel.: +55 35 38291916; fax: +55 35 38291100. E-mail address: [email protected]fla.br (C.F. Silva). 0956-053X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.wasman.2010.08.015

(Navarro et al., 2000). The vinasse can be stored in decantation tanks within the manufacturing unit, used as a fertilizer, or released into a water course. The major problem relating to acceptable methods of disposal of vinasse derived from bio-ethanol and cachaça production is the associated need to protect the environment from the extremely large quantities of vinasse that are generated by the industrial production of fuel alcohol. The vinasse contain carbon compounds and nitrogen assumable by the microorganisms and is widely available, therefore it could be used for production of microbial biomass. This biomass could be used as a protein supplement (single cell protein – SCP) for animal feed (Pandey, 2003; Ugalde and Castrillo, 2005; Zheng et al., 2005). Application of the residue from these industries as an abundant and low-cost raw material in the production of food grade yeast, for example, would not only be economically viable but would also solve problems caused by the accumulation of toxic waste in bioethanol and cachaça-producing areas. Vinasse also offers the added advantage that it is relatively free of toxins and fermentation inhibitors (Bekatorou et al., 2006). SCP is normally considered to be a valuable source of protein, but it also contains nucleic acids, carbohydrates, cell wall material, lipids, minerals and vitamins (Ugalde and Castrillo, 2005). The bioconversion by fungi of residues originating from distilleries offers two extra advantages, namely, a potentially toxic effluent is substantially purified and the resulting product has useful applications (Friedrich, 2004). Saccharomyces cerevisiae and

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Candida parapsilosis are the principal microorganisms used in fermentation processes associated with the production of bio-ethanol and spirit alcohol, and as such they are well-adapted to harsh and highly competitive fermentation conditions (Schwan et al., 2001; Bernardi et al., 2008). Additionally, these microorganisms retain their viability and grow well on solid and liquid agro-industrial residues. The aims of the present study were to evaluate the biomass production by yeasts isolated from cachaça and bio-ethanol industries utilizing vinasse as substrate, and to investigate the effects of culture conditions on the production of microbial biomass from vinasse, and to examine the nutritional quality of the product generated.

Table 1 Conditions of variables at different levels using Plackett–Burman experimental design for biomass production from vinasse. S. No.

Variables

Low level ( )

High level (+)

X1 X2 X3 X4 X5 X6 X7

Yeast extract (%) Glucose (%) Peptone (%) Potassium phosphate (%) Vinasse added (v/v) pH Incubation temperature (°C)

1.0 2.0 1.0 0.05 10 3 28

3.0 4.0 3.0 0.2 50 5 36

percentage from the content of final biomass. The dry mass of yeast biomass was measured after drying to constant weight at 105 °C.

2. Materials and methods 2.1. Microorganisms

2.4. Evaluation of the relative importance of the nutritional and physicochemical parameters of the culture medium

Microorganisms were obtained from the Collection of the Laboratory of Physiology and Microbial Genetics of the Biology Department at Federal University of Lavras, Lavras, MG, Brazil. The isolates involved were: (i) S. cerevisiae (UFLACA15, UFLACA76, UFLACA93, UFLACA155) and C. parapsilosis (UFLACA271) originating from cachaça-producing units; and (ii) S. cerevisiae (PE2, CAT1, VR1) originating from fuel alcohol-producing plants. Each microorganism was inoculated into 250 mL of culture broth containing different concentrations of glucose (2% or 4%), yeast extract (1% or 3%), peptone (1% or 3%), potassium phosphate (0.05% or 0.2%) and vinasse (10% or 50% v/v). The pH of the culture was adjusted to 3.0 or 5.0 and incubation was carried out at 28 or 36 °C for until 192 h. During this period, samples were collected daily for chemical and microbiological analyses.

The experimental design was based on the model proposed by Plackett and Burman (1946). A total of 10 variables were tested, seven of which were associated with physicochemical parameters (concentration in the medium of yeast extract, glucose, peptone and potassium phosphate, amount of vinasse added, pH of medium, and incubation temperature) and three were associated with inert variables that were used for the calculation of standard errors. The Table 1 shows the maximal and minimal levels used for each variable tested. Following cultivation of the fungal isolates, the characteristics of the biomass obtained (dry mass, nitrogen content and nucleic acid content) were evaluated, and the production of microbial biomass (O) and productivity (Y = O/h for cultivation) of the cultures determined.

2.2. Origin and analysis of vinasse

2.5. Statistical analyses

Vinasse was supplied by a traditional cachaça-producing unit, located in the Southern area of the State of Minas Gerais, during the 2008 season. Vinasse was cooled and subsequently frozen until required for the experiments. When appropriate, the desired volume of vinasse was defrosted, filtered and autoclaved at 121 °C for 15 min. Vinasse was added to the culture medium 8 h after inoculation of the microorganisms. Prior to the experiments, the physicochemical characteristics of vinasse were determined according to standard procedures. The parameters analysed were: glucose, sucrose and total sugar (Nelson, 1944), cellulose, hemicellulose, and lignin (Van Soest and Wine, 1968), crude fibre (Van der Kamer and Van Ginkel, 1952), total carotenoids, chlorophyll and nitrate (Cataldo et al., 1975), soluble and total pectin (Bitter and Muir, 1962), tannins and solubility (Deshpande et al., 1986), water content and ash residue (Instituto Adolfo Lutz, 1985), ether extract and protein (AOAC, 1970), total titrated acidity (AOAC, 2000), pH, chemical oxygen demand COD, biochemical oxygen demand BOD, dissolved oxygen and total solids (APHA, 1992).

The effects of each independent variable over the dependent variables were determined using equation Ep = [ep(ef)
n1/2]/2, in which ep(ef) represents {[(U1)2 + (U2)2 + (U3)2 . . . (Uq)2]/q}1/2 where P Ui is given by ( IV  Y)/n, q is the number of inert variables (IV), Y is biomass, nitrogen content or nucleic acids content, and n is the number of assays. The threshold for statistical significance was established at P 6 0.05.

2.3. Analysis of biomass produced Cell viability of the culture was analysed using methylene blue and a Neubauer-counting chamber (Lee et al., 1981). The total number of colony forming units (cfu) in the culture was determined every 24 h. The nitrogen content was estimated according to the Kjeldahl method (APHA, 1992). Microbial DNA and RNA were extracted using the procedure of Van Elsas et al. (1997) and quantified spectrometrically at 230 nm using a Nanodrop ND 1000 spectrometer. The absorbances obtained were converted in

3. Results and discussion 3.1. Effect of variables tested on production and nitrogen content in microbial biomass Physicochemical analysis of vinasse samples (Table 2) revealed the presence of low levels of total sugars, mineral components (ash), ether extract and protein (0.18%, 0.24%, 0.04% and 0.33%, respectively), indicating that it would be necessary to add glucose, yeast extract and potassium phosphate to the culture medium. The most abundant substances in the vinasse samples were nitrate, total pectin and tannin, and these constituents could hamper microbial growth either because they are not readily metabolised or are deleterious to the assimilation of proteins, as is the case for tannins. Assays were conducted according to the Plackett and Burman (1946) experimental model in which the culture parameters were varied simultaneously (Table 3). In this manner, the influence of these parameters on the production and productivity of yeast biomass could be estimated. The production of biomass varied between 0.7 and 15.0 g L 1, depending on the culture conditions, suggesting that the vinasse could be recycled in order to maximize

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C.F. Silva et al. / Waste Management 31 (2011) 108–114 Table 2 Physicochemical characteristics of fresh vinasse added to the culture medium. Parameter

Value

Total sugars (%) Glucose (%) Sucrose (%) Solubility (%) Water content (%) Protein (%) Ash (%) Crude fibre (%) Ether extract (%) Total carotenoids (mg 100 g 1) Chlorophyll (mg 100 g 1) Nitrate (g 100 g 1) Total pectin (mg 100 g 1) Soluble pectin (mg 100 g 1) Tannin (mg 100 g 1) Cellulose (mg 100 g 1) Lignin (mg 100 g 1) Hemicelluloses (mg 100 g 1) Total acidity titre (v/v) Chemical oxygen demand (g L 1) Biochemical oxygen demand (g L Dissolved oxygen (mg L 1) Total solids (g L 1) pH

0.18 0.14 0.04 6.51 98.0 0.33 0.24 0 0.04 0.003 0.32 31.0 91.4 5.9 80.47 0 0 0 0.4 57.5 1.9 1.0 24.56 7.27

1

)

the use of substrate during the production of microbial biomass (Table 3). Following studies on Candida utilis grown on sugar molasses, Lee and Kim (2001) observed that when the initial concentration of glucose was high (10%) productivity increased although the biomass decreased. In the present study, the correlation between glucose concentration and biomass production was not observed since the largest production of biomass for some isolates, in some assays, occurred in medium containing a high level of glucose (i.e. 4%) and other assays to higher production biomass occurred at lower glucose concentrations (e.g. isolated UFLACA 93 assays 1 and 7). Regarding the productivities of the isolates studied, the majority varied within the range of 0.004–0.06 g L 1 h 1, but those of VR1 and PE2 were remarkably superior and varied in the ranges 0.04–0.1 g L 1 h 1 and 0.03–0.1 g L 1 h 1, respectively (Table 3). With regard to VR1, the assay conditions that were most productive were 9 and 12, which contained similar and lower concentrations of glucose (2%), yeast extract (1%), peptone (1%) and potassium phosphate (0.05%), but differed with respect to vinasse (50% and 10% v/v, respectively), pH (5.0 and 3.0, respectively)

and temperature (36 and 28 °C, respectively). In contrast, isolate PE2 was not influenced by any of these variables, and maximum productivity was achieved independent of the concentration of vinasse employed. It may be concluded from these results that interactions within the incubation medium do not generally influence productivity. It has previously been demonstrated that glucose, potassium phosphate, yeast extract and temperature directly affect the utilization and growth of fungal cultures on vinasse (Friedrich, 2004; Enjalbert and Whiteway, 2005; Ugalde and Castrillo, 2005). However, it was observed here that these parameters showed either negative or weak effect on biomass production and nitrogen content (Table 4). The fermentation media contained a readily available carbon source (glucose) that could improve the utilization of vinasse and fungal growth, there were no significant differences between seven isolates with respect the production of biomass (Table 4), except the isolate CAT1, which showed weak negative effect on biomass production ( 0.2). Biomass production of isolated CAT1 also suffered weak significant effect of variable concentration of vinasse and yeast extract, pH and temperature. Biomass production of others strains were significant weaknesses in the concentration of peptone, vinasse, pH and temperature (Table 4). Concentrations of peptone, vinasse and incubation temperature had significant effect on nitrogen content of only isolated UFLACA15 (positive effect), UFLACA93 (negative effect) and UFLACA76 (negative effect), respectively. The other isolates did not show the content of nitrogen influenced by levels of variables tested. Generally, vinasse, peptone, pH and temperature presented a statistically significant negative effect, although these parameters could exert a positive influence depending on the isolate (Table 4). It is known that temperature can affect the growth rate, oxygen diffusion and metabolic pattern of cultures (Ugalde and Castrillo, 2005). 3.2. Nutritional quality of biomass and yeast cell viability Table 5 shows the characteristics of the biomass obtained. In order to serve as a health-conserving animal feed supplement, the microbial biomass must fulfil certain nutritional prerequisites including high contents of nutritional compounds (total nitrogen, for example) and low levels of anti-nutritional compounds (i.e. nucleic acid). With respect to the latter, ingested nucleic acids are degraded to purine and pyrimidine bases, and after breakdown of purine bases produces uric acid leading to gout-like manifestations and calculi in the urinary tract (Ugalde and Castrillo, 2005). The content of nucleic acids found was higher than those normally

Table 3 Results of production of microbial biomass and productivity determined until 192 h incubation of eight yeasts isolates using the experimental design Plackett–Burman. Maximum productivity values are bolded. Assay

Variables X1

1 2 3 4 5 6 7 8 9 10 11 12

+ + + + +

X2

Biomass Production (O) and Productivity (Y) X3

+ + + + + +

+ +

X4

X5

X6

X7

+ +

+ +

+ +

+ + +

+ + + +

+ + + + +

+ + + + + +

+ + + + +

Yeasts UFLACA15

UFLACA76

UFLACA93

UFLACA155

UFLACA271

PE2

O

Y

O

Y

O

Y

O

Y

O

Y

O

Y

O

Y

O

Y

3.9 6.1 5.4 3.4 4.8 1.2 0.7 6.1 3.8 6.1 5.6 6.6

0.02 0.04 0.03 0.02 0.03 0.007 0.004 0.04 0.02 0.04 0.03 0.04

6.9 5.0 6.5 1.7 3.9 5.4 3.2 6.5 3.0 5.3 4.5 5.6

0.04 0.03 0.04 0.01 0.02 0.03 0.02 0.04 0.02 0.03 0.03 0.03

8.7 6.4 7.9 5.4 7.9 7.1 11.4 7.6 8.3 7.0 4.2 5.3

0.04 0.03 0.04 0.03 0.04 0.04 0.06 0.04 0.04 0.04 0.02 0.03

4.3 3.5 6.0 6.3 7.2 3.8 5.2 0.9 5.7 8.5 6.3 5.5

0.02 0.02 0.03 0.03 0.04 0.02 0.03 0.005 0.03 0.04 0.03 0.03

7.8 4.8 5.8 7.3 5.3 7.5 4.6 8.2 5.6 6.8 4.4 4.2

0.04 0.03 0.03 0.04 0.03 0.04 0.03 0.04 0.03 0.03 0.02 0.02

12.3 9.0 4.9 14.3 6.9 8.6 9.0 13.2 8.3 9.1 10.2 8.9

0.09 0.06 0.03 0.1 0.05 0.06 0.06 0.09 0.06 0.06 0.07 0.06

6.1 7.4 7.1 6.1 7.2 8.8 8.7 6.9 13.8 9.7 10.1 15.0

0.04 0.05 0.05 0.04 0.05 0.06 0.06 0.05 0.1 0.07 0.07 0.1

4.6 5.0 5.9 2.9 5.2 – 2.2 5.7 4.3 2.7 2.2 3.4

0.03 0.03 0.04 0.02 0.04 – 0.02 0.04 0.03 0.02 0.02 0.02

VR1

CAT1

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C.F. Silva et al. / Waste Management 31 (2011) 108–114 Table 4 Effect of parameters tested on biomass production (g L UFLACA15

1

) and nitrogen content (%) during cultivation of eight yeasts isolates in the both levels of studies factors.

UFLACA76

Biomass Yeast extract Glucose Peptone KH2PO4 Vinasse pH Temperature

0.11 0.19 0.51* 0.02 0.15 0.04 0.51*

0.04 0.01 0.12 0.13 0.46*

Nitrogen Yeast extract Glucose Peptone KH2PO4 Vinasse pH Temperature

0.3 0.33 0.57* 0.07 0.17 0.03 0.1

3.45 0.22 0.02 0.18 0.55 0.22 1.15*

UFLACA93

0.16 0.47*

UFLACA155

UFLACA271

PE2

VR1

CAT1

0.1 0.1 0.38 0.26 0.29 0.18 0.02

0.19 0.01 0.44* 0.09 0.21 0.02 0.37*

0.29 0.31 0.43* 0.1 0.03 0.26 0.002

0.25 0.8 0.42 0.3 0.3 0.8 0.2

0.7 0.5 0.8 0.4 0.3 1.7* 0.32

0.2* 0.2* 0.08 0.02 0.25* 0.2* 0.65*

0.3 0.2 0.07 0.1 0.7*

0.03 1.2 0.23 0.2 0.4 0.4 0.03

0.3 0.6 0.6 0.4 0.06 0.03 0.4

0.4 0.4 0.23 0.3 0.5 0.17 0.4

0.02 0.1 0.5 0.9 0.18 0.4 0.9

0.5 1.25 0.75 1.7 0.5 1.4 2.3

0.07 0.17

Significant at P 6 0.05.

Table 5 Characteristics of the microbial biomass determined until 192 h incubation of eight yeasts isolates. Assays

UFLA CA 15a

UFLA CA76

Nc

NAd

Nc

NAd

Nc

NAd

Nc

67 63 79 >90 >90 >90 >90 >90 >90 76 89 61

9.26 9.85 9.79 9.43 10.6 8.56 8.75 9.93 8.83 9.1 7.6 9.81

9 37 21 >90 29 34 32 16 32 21 7 13

9.84 9 9.17 9.12 9.03 9.7 8.7 9.24 8.48 9.75 9.37 9.78

17 31 29 36 68 31 33 32 35 50 >90 26

9.70 7.84 9.23 9.79 9.15 7.15 7.71 8.53 10.95 9.03 8.10 8.41

% dry matter 1 9.31 2 8.81 3 9.2 4 9.9 5 8.8 6 9.10 7 9.36 8 9.21 9 8.8 10 9.4 11 8.13 12 8.73 a b c d

a

UFLA CA93

a

UFLA CA271b

PE2

NAd

Nc

NAd

Nc

NAd

Nc

NAd

Nc

NAd

27 39 30 33 26 57 42 >90 37 21 31 29

9.69 9.03 9.35 9.43 8.1 8.6 8.6 9.1 8.84 8.92 7.39 8.75

16 82 26 42 39 42 63 25 62 23 50 30

9.62 9.14 8.87 8.97 8.38 9.52 8.21 9.03 8.14 9.05 8.27 9.72

29 20 58 21 34 33 20 25 49 24 39 26

9.75 9.56 9.41 9.6 9.26 9.7 8.57 9.21 9.02 6.82 8.41 9.95

28 23 30 50 36 33 30 19 21 30 22 14

10.3 9.1 9.4 8.1 8.53 – 7.71 8.33 7.13 8.56 7.56 7.35

41.4 73.1 23.0 50.7 16.6 – 138.5 16.2 92.2 61.9 132.6 40.0

UFLA CA155

a

a

VR1

a

CAT1

a

Saccharomyces cerevisiae isolate. Candida parapsilosis isolate. N, nitrogen content. NA, nucleic acid content.

acceptable for animal supplements (610% dry mass). The mean nitrogen values observed (9%) were equivalent to 56.25% of protein (employing a correction factor of 6.25). These results were similar or superior to the levels of protein content that have been determined for microbial biomass grown in different substrates and are values which is suitable for dietary supplementation (Choi et al., 2002; Paul et al., 2002), hence, vinasse may be considered an adequate substrate for the production of SCP. Ziino et al. (1999) reported that biomasses of Saccharomyces and Candida spp. produced by fermentation of orange residues exhibited a high protein content (up to 50% dry mass), whereas those of other microbial species (Geotrichum candidum for example) presented protein values that were lower than 40% dry mass. 3.3. Microscopy evaluation of yeasts isolates during growth on vinasse All isolates tested were able to use vinasse as substrate, although there were differences regarding the final biomass, which may be associated with the origin of the isolates (bio-ethanol industry or cachaça units). The total population of isolate PE2 was 104  107 cfu mL 1 after 144 h incubation under the cultivation conditions of assay 10 (high levels of yeast extract, potassium phosphate, pH and temperature, low levels of others

factors) (Fig. 1). The total population of isolate VR1 was 2.2-fold greater than the inoculum under the conditions of assays 3 (high levels of glucose, peptone and pH, low levels of others factors) and 8 (high levels of peptone, potassium phosphate, vinasse and pH, low levels of others factors) (Fig. 2). The superior growth and higher tolerance to stress exhibited by isolates PE2 and VR1 may be explained by their origin, in that these strains were derived from the fermentation tanks of a fuel alcohol production plant. Owing to the greater tolerance to the stress factors, the consumption of carbon by these isolates was also greater compared with the other yeasts isolates. The isolates PE2 and VR1 were able to consume 92% and 95.5%, respectively, of the reducing sugars present in the medium in the first 24 h (Figs. 1 and 2). The isolate UFLACA15, from a cachaça-producing unit, was highly adapted to the cultures conditions since the maximum population was 31  107 cfu mL 1 recorded after 72 h incubation under the cultivation conditions of assay 5 (high levels of yeast extract, glucose, potassium phosphate, vinasse and pH, low levels of others factors) (data not shown). The fast growth observed until 72 h did not reflect a greater biomass conversion at the end of the cultivation period, during which the consumption of carbon was only 17.5% (data not show).

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Fig. 1. Total population (gray and black bars) of isolate PE2 cultivated during 24 and 144 h under 12 different assay conditions as defined by experimental model by Plackett– Burman. Percentage of reducing sugar consumption (white bars). Variables tested 1–12 (see Table 3).

Fig. 2. Total population (gray and black bars) of isolate VR1 cultivated during 24 and 144 h under 12 different assay conditions as defined by experimental model described Plackett–Burman. Percentage of reducing sugar consumption (white bars). Variables tested 1–12 (see Table 3).

In contrast with isolate UFLACA15, the total population of UFLACA271 after 72 h of cultivation was lower than the initial inoculum and growth was observed only after 144 h (data not shown). For this reason, the culture period for this isolate was extended to 192 h, at which time the total population had increased to 1.38  1010 cfu mL 1 under assay 4 (high levels of yeast extract, peptone, vinasse and temperature, low levels of others factors). In this respect, isolate UFLACA271 exhibited the highest cell growth under the various culture conditions employed of all of the isolates studied, although the production of biomass and productivity were similar to the other isolates originated from cachaça-producing units.

The physicochemical parameters of the eight fungal isolates studied had no marked influence on biomass production or productivity. However, some morphological differences were detected following microscopic investigation of the isolates during the culture period, the most prominent of which were the decrease in cell size and the presence of cluster-like cell agglomerates, multiplesprouting spots and pseudo hyphae observed after 24 h incubation (Fig. 3). Alterations in the shape of S. cerevisiae cells (from oval to filamentous) are correlated not only with nutritional and physical conditions, but also with the accumulation of products resulting from the metabolism of amino acids that occurs when nutrients are limited (Ceccato-Antonini, 2008). According to Pillai et al.

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Fig. 3. Optical (A) and scanning electron (B, C and D) photomicrographs of isolate CAT1 grown under the conditions of assay 6 (3% yeast extract, 4% glucose, 3% peptone, 10% v/v vinasse, 0.05% potassium phosphate at 36 °C and pH 5) showing the morphological alterations that occurred during the total culture period.

(2003), yeast cells are able to survive adverse conditions, including extreme temperature and nutrient depletion, by activating silent genes. For example, the non-essential Rpb4 subunit of RNA polymerase II is required for the survival of yeast cells and, in its absence, the cells acquire the appearance of pseudo-mycelia. Different cell shapes exhibit dissimilar physiological characteristics, and such versatility may facilitate fungal adaptation to environmental fluctuations. Indeed, many species of hemiascomycete yeasts (S. cerevisiae, Candida albicans, Candida glabrata, Candida tropicalis and C. parapsilosis) can switch frequently and reversibly between ‘states’ or ‘phases’ (Malagnac and Silar, 2003). Extremes of pH and temperature can, in certain species of Candida, induce the activation of genes that promote the development of filaments (d’Enfert and Hube, 2007). Although the growth of isolates was not synchronised in the present study, cells were observed to sprout or develop pseudomycelia (Fig. 3) indicating that they were entering the stationary phase. Enjalbert and Whiteway (2005) also observed that at the beginning of the stationary phase Candida cells were more competent for the formation of hyphae, and samples containing such cells produced more hyphae. 4. Conclusions The experimental design Plackett–Burman allowed verifying the variables that had any effect on production and quality of the yeast biomass produced. Among the variables tested, the concentration of peptone, vinasse and temperature had positive effect on biomass production, while peptone was the only variable significantly and positively influencing the nitrogen content. Analysis of the nutritional quality of biomass suggests that it could be a source of supplemental protein for animal feed since the nitrogen concentration was on average 9% d.m. Thus our results suggested that the vinasse could be used to produce SCP because the addition of car-

bon and nitrogen sources in the culture medium were relatively low and that could help to minimize the environmental pollution caused by the industrial by-product. Of the S. cerevisiae isolates tested, two (VR1 and PE2) originating from alcohol fuel-producing plants were identified as offering the best potential for the industrial production of SCP in medium containing 50% (v/v) of vinasse. Acknowledgements The authors are grateful to Dr. Maria Isabel Rodrigues and Dr. Carla da Silva Ávila for assistance with the statistical analyses. CFS wishes to thank FAPEMIG for a postdoctoral grant and for financial support. References APHA, 1992. American Public Health Association. Standard Methods for the Examination of Water and Waste Water, 18th ed. American Public Health Association, Washington, USA. Anuário estatístico da Agroenergia, 2009. Ministério da Agricultura, pecuária e Abastecimento. Secretaria de Produção e Agroenergia, Brasília. 161p. ABNT, 2004. Associação Brasileira de Normas Técnicas. NBR 10.004. (accessed 24.09.2009). AOAC, 1970. Association of Official Analytical Chemists. Official Methods of Analysis of the Association of Official Analytical Chemists, 11th ed. AOAC, Washington, USA. 1015p. AOAC, 2000. Association of Official Analytical Chemists, Official Methods of Analysis of the Association of Official Analytical Chemists, 17th ed., vol 2. AOAC, Gaithersburg, USA, pp. 915–922. Bekatorou, A., Psarianos, C., Koutinas, A., 2006. Production of food grade yeasts. Food Technology and Biotechnology 44 (3), 407–415. Bernardi, T.L., Pereira, G.V.M., Cardoso, P.G., Dias, E.S., Schwan, R.F., 2008. Saccharomyces cerevisiae strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR, PFGE and mtDNA-RFLP). World Journal of Microbiology and Biotechnology 24 (11), 2705–2712. Bitter, V., Muir, H.M., 1962. Modified uronic acid carbazole reaction. Analytical Biochemistry 4 (4), 330–334.

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