Environmental Technology

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Cheese Whey Permeate Fermentation by

Kluyveromyces lactis: a Combined Approach to
Wastewater Treatment and Bioethanol Production

Fábio Coelho Sampaio, Janaína Teles de Faria, Milena Fernandes da Silva,

Ricardo Pinheiro de Souza Oliveira & Attilio Converti

To cite this article: Fábio Coelho Sampaio, Janaína Teles de Faria, Milena Fernandes da Silva,
Ricardo Pinheiro de Souza Oliveira & Attilio Converti (2019): Cheese Whey Permeate Fermentation
by Kluyveromyces�lactis: a Combined Approach to Wastewater Treatment and Bioethanol
Production, Environmental Technology, DOI: 10.1080/09593330.2019.1604813

To link to this article: https://doi.org/10.1080/09593330.2019.1604813

Accepted author version posted online: 06

Apr 2019.

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Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis Group

Journal: Environmental Technology

DOI: 10.1080/09593330.2019.1604813

Cheese Whey Permeate Fermentation by Kluyveromyces lactis: a Combined

Approach to Wastewater Treatment and Bioethanol Production

Fábio Coelho Sampaioa, Janaína Teles de Fariab, Milena Fernandes da Silvac, Ricardo

Pinheiro de Souza Oliveirad, and Attilio Convertie,*

a
Department of Microbiology, Federal University of Viçosa, Viçosa, MG, Brazil
b
Agricultural Sciences Institute, Federal University of Minas Gerais, Montes Claros, MG, Brazil
c
Bioscience Center, Federal University of Pernambuco, Recife, PE, Brazil
d
Department of Biochemical and Pharmaceutical Technology, University of São Paulo, SP, Brazil
e
Department of Civil, Chemical and Environmental Engineering, Genoa University, Genoa, Italy

______________
*
Corresponding author.

Attilio Converti, Department of Civil, Chemical and Environmental Engineering, Pole of Chemical

Engineering, Genoa University, Via Opera Pia 15, I-16145 Genoa, Italy, Phone: +39-3292104448;

Fax: +39-010-3532586; E-mail address: converti@unige.it (A. Converti).

1
Cheese Whey Permeate Fermentation by Kluyveromyces lactis: a Combined

Approach to Wastewater Treatment and Bioethanol Production

Cheese whey is a dairy industry by-product responsible for serious environmental problems. Its

fermentation would allow reducing its environmental impact and producing, at the same time, high-

value products, hence ensuring cleaner production. Batch fermentations of cheese whey permeate,

either as such or 1.5-fold or twice-concentrated, by Kluyveromyces lactis CBS2359 were performed

in flasks with or without agitation to select the best conditions to produce simultaneously ethanol and

biomass with high β-galactosidase activity. In shake cultures, the highest ethanol concentration (15.0

g.L-1), yield on consumed lactose (0.47 g.g-1) and productivity (0.31 g.L-1.h-1), were obtained on

cheese whey permeate as such, corresponding to 87.4% fermentation efficiency, but β-galactosidase

activity was disappointing (449.3-680.0 U.g-1). In static cultures on twice-concentrated whey

permeate, despite a decrease in fermentation efficiency and yield, ethanol production increased by

48% and β-galactosidase activity by no less than 209-367%. Therefore, cheese whey should be

considered an alternative feedstock rather than an undesirable dairy industry by-product.

Keywords: Cheese whey permeate; Bioethanol; β-Galactosidase; Kluyveromyces lactis; Food

industry wastewater.

2
1 Introduction

Cheese whey permeate is a by-product of dairy industry formed during the coagulation of milk

casein that retains about 55% of milk nutrients and may be used as an alternative source of added

value compounds [1-5]. It is composed mainly of water and approximately (w/v) 4.5-5.0% lactose,

0.6-0.8% soluble proteins, 0.4-0.5% lipids, among other components [2,6,7]. Lactose, which is

responsible for the high biological (30–50 g L-1) and chemical (60–80 g L-1) oxygen demands of

cheese whey [1], may be used as a carbon source in different bioprocesses, thereby greatly reducing

the serious environmental problems related to its disposal. Therefore, the profitable recovery of

whey permeate is currently a hot investigation topic, which represents a big challenge due to the

huge amount produced worldwide [8].

Recently, many studies have been carried out on the alternative use of such an agro-

industrial by-product to make a proper control and a complete valorization of dairy industry waste

[9-11]. To give only a few examples, cheese whey formulated media were successfully employed to

produce, besides bioethanol [1,12-15], biohydrogen [16], prebiotics [17,18], bacteriocin [19],

polyhydroxy acid [20], hyaluronic acid [21] and butanol [22]. Deproteinized cheese whey was also

investigated as an alternative medium to produce the yeast Kluyveromyces lactis as single-cell

protein [23].

Among the microorganisms able to metabolize lactose, K. lactis stands out for its ability to

metabolize this disaccharide through the activities of a lactose permease and a β-galactosidase (β-

gal; β-D-galactosidegalactohydrolase, EC 3.2.1.23). β-gal, which is one of the most important

enzymes used in food processing, has nutritional, technological, and environmental applications

[24]. In this regard, because of its effectiveness, K. lactis β-gal has recently gained interest

especially in the enzymatic synthesis of compounds of industrial relevance [25,26].

Despite being designated as Crabtree negative species, i.e., predominantly respiratory [27],

K. lactis has the potential to get energy via the respirofermentative metabolism in response to

3
oxygen limitation [27,28], while it is unable to grow under strict anaerobic conditions [29]. This

Generally Recognized as Safe (GRAS) yeast [30] has become an alternative to the traditional

Saccharomyces cerevisiae owing to its industrial potential [2,27,31].

Although hardly economically competitive with the currently established fermentation

processes using sugarcane, cornstarch or lignocellulosic biomass as substrates [1], whey

fermentation has received wide attention nowadays as a way to obtain high value products

[8,12,13,15,16,32-35], because it allows combining in a valuable production strategy several

advantages, among which are a) the treatment of a polluting by-product, b) the production of

bioethanol and c) the production of yeast biomass with high β-gal activity.

Based on this background, the aim of this study was to investigate the ability of K. lactis to

ferment cheese whey permeate either as such or concentrated, in terms of ethanol production and β-

gal activity. To this purpose, batch cultures were performed in flasks either with or without

agitation. The main novelty of this study relies on the effort to reduce the impact related to the

release of huge amounts of cheese whey to the environment, by exploiting it as a potential

alternative feedstock for the simultaneous productions of bioethanol and yeast biomass with high β-

gal activity.

2 Materials and methods

2.1 Microorganism and cultivation medium

The Kluyveromyces lactis CBS2359 strain, belonging to the collection of the Federal University of

Viçosa (UFV), Viçosa, MG, Brazil, was used in all the experiments. It was maintained in Petri

dishes on agarized Yeast Protein Dextrose medium (10 g L-1 yeast extract, 20 g L-1 peptone, 20 g L-

1
D-glucose and 15 g L-1 agar). After the inoculum, dishes were incubated at 331°C for 48 h and

stored at 4°C in refrigerator. The culture was subcultured every two weeks.

4
 The nutrient medium was cheese whey permeate (CW) obtained from Ars Food (Varese

Ligure, Italy). Typically, 1.0 L-aliquots were stored at -10oC in freezer. After defrosting at 60oC and

homogenizing, CW pH was adjusted to 5.0. To obtain media with higher lactose content, CW was

1.5-fold or twice-concentrated at 75oC under vacuum, using a rotary vacuum evaporator, model

Laborota 4000 (Heidolph, Schwabach, Germany). CW and concentrated CWs were autoclaved at

121oC for 15 min to coagulate whey proteins, kept at room temperature for 14-16 h and then filtered

through filter paper. Supernatants were collected and autoclaved again (121oC for 15 min). After

these treatments, the protein content in CW determined by the micro-Kjeldahl method was 0.86%

(w/v).

2.2 Inoculum preparation and culture conditions

To prepare the inoculum, loopfuls of cells grown for 48 h at 33°C were transferred from dishes to

250 mL-Erlenmeyer flasks containing 50 mL of CW as such. Flasks were maintained at the same

temperature under agitation at 180 rpm. After 16–18 h of incubation, cells were collected by

centrifugation at 2000 g for 5 min, washed twice with sterile water and used for the inoculum at a

dry biomass concentration in the range 21-28 mg L-1. Batch cultures were conducted at 331°C,

either with agitation at 180 rpm or not, in 250 mL-Erlenmeyer flasks containing 50 mL either of

CW as such or concentrated CWs. Cell concentration was monitored at different times along the

entire fermentations, and all its values collected at the exponential growth phases were used to

estimate the specific growth rates as explained in the next section. By observing the trend of each

growth curve, we then established the time intervals 3-10, 11-48 and 49-96 h as representative of

the exponential, early stationary and late stationary growth phases in shake cultures, and 24-48, 49-

72 and 73-96 h in the static ones. All the fermentations and experimental measurements were

performed in triplicate.

2.3 Determinations of biomass concentration and specific growth rate

5
Biomass concentration either in the fermentation broth or the inoculum suspension was determined

by optical density (OD) measurements at 600 nm using a UV-Vis spectrophotometer, model

Lambda 25 (Perkin Elmer, Milan, Italy), after subtraction of medium absorbance. Flasks for static

cultures were vigorously shaken only before sampling to prevent settling of solids and then

heterogeneity in samples. A calibration curve (1 OD = 0.212 g L-1 biomass) was used to relate OD

with cell dry weight determined after drying at 1001°C. The specific growth rate was determined

by linear regression as the slope of the plot of lnOD at 600 nm versus time at the exponential

growth phase.

2.4 Analytical methods

Aliquots of cultures were centrifuged at 2000 g for 5 min, and supernatants used for determination

of lactose and ethanol concentrations. These metabolites were quantified by high performance

liquid chromatography, using a HPLC, model 1100 (Hewlett Packard, Palo Alto, CA, USA),

equipped with a refractive index detector, model HP 1047 A, and a 7.7 x 300 mm Hi-Plex H

column, model PL1170-6830 (Supelco, Agilent, Santa Clara, CA, USA). A 0.01 N H2SO4 solution

was used as the eluent at a flow rate of 0.5 mL min-1, and the analyses were carried out at 50oC. β-

Galactosidase (β-gal) activity was determined as previously described by Gietz et al. [36]. One unit

(U) of β-gal activity was defined as the amount of enzyme able to catalyze the release of 1.0 µmol

of o-nitrophenol per minute. The enzyme activity detected in a 0.5 mL-sample of cell suspension

was finally expressed as specific enzyme activity referred to one gram of dry cell mass (U g−1).

2.5 Fermentation parameters

The yield of biomass on consumed lactose (YX/S), expressed in g biomass g-1 lactose, was

determined according to the equation:

𝑋𝑓 − 𝑋0
𝑌𝑋/𝑆 = (1)
𝑆0 − 𝑆𝑓

6
where Xf and X0 are the dry cell mass concentrations at the end and beginning of cultivations (g L-1),

and Sf and S0 the corresponding lactose concentrations (g L-1).

The yield of ethanol on produced biomass (YE/X), expressed in g ethanol g-1 biomass, or of

ethanol on consumed lactose (YE/S), expressed in g ethanol g-1 lactose, was determined according to

the equations:

𝐸𝑓 𝐸𝑓 (2)
𝑌𝐸/𝑋 = and 𝑌𝐸/𝑆 =
𝑋0 − 𝑋𝑓 𝑆0 − 𝑆𝑓

where Ef is the ethanol concentration (g L-1) at the end of cultivations.

The coefficient of correlation between the specific β-gal activity (xi), expressed in U g-1

biomass, and YE/S (yi) was calculated according to the equation [37]:

∑(𝑥𝑓 −𝑥̅ ) (𝑦𝑖 −𝑦

̅)
𝑟= (3)
√∑(𝑥𝑖 −𝑥̅ )2 ∑(𝑦𝑖 −𝑦
̅)2

where 𝑥̅ e 𝑦̅ are the mean values of the same variables.

The mean ethanol volumetric productivity (QE, g L-1 h-1) and specific productivity (qE, g

ethanol g-1 biomass h-1) were defined as:

𝐸𝑓 𝑄𝐸 (4)
𝑄𝐸 = and 𝑞𝐸 =
𝑡 𝑋𝑓 − 𝑋0

where t is the cultivation time (h).

The fermentation efficiency (FE, %) was calculated as:

7
 𝑌𝐸/𝑆 (5)
𝐹𝐸 = × 100
𝑌𝑇

where YT is the theoretical ethanol yield, corresponding to 0.538 g ethanol g-1 lactose [15].

2.6 Statistical analysis

Effects were compared by the Tukey’s test when necessary, using the Statistical Analysis System

software, version 9.1 (SAS Institute Inc., Cary, NC, USA). Significance of variations was presented

in the form of probability values (p  0.05). Average values labelled with the same letters did not

exhibit any statistically significant difference between them according to the Tukey’s test (p >

0.05).

3 Results and discussion

The importance of sugar and oxygen concentrations in determining the metabolic preference of

yeasts, and consequently ethanol formation under a given growth condition, seems to be a general

feature [27]. However, whey contains proteins, lipids, mineral salts and other compounds in

addition to lactose [7], which may strongly influence yeast growth and lactose fermentation.

Therefore, the batch fermentative behavior of Kluyveromyces lactis was investigated in cheese

whey permeate either as such (CW1) or 1.5-fold (CW2) or twice (CW3) concentrated, with or

without agitation, while strict anaerobiosis was not tested because of the well-known inability of

this yeast to grow under these conditions [29].

3.1 Biomass concentration and specific growth rate

8
As shown in Fig. 1, both K. lactis specific growth rate () and biomass concentration (X) increased

with starting lactose concentration (So). After 24 h of shake cultures, X values (3.01 and 3.25 g L-1)

obtained in CW2 and CW3, which contained 55.0 and 75.0 g L-1 lactose, respectively, were not

statistically significantly different between them according to the Tukey’s test (p > 0.05), but were

84 and 70% higher, respectively, than that obtained in cheese whey permeate as such containing

38.0 g L-1 lactose (Fig. 1B). Similarly,  was in CW2 (0.461 h-1) and CW3 (0.488 h-1) about 15-

21% higher than in CW1 (Fig. 1A). A qualitatively similar effect was observed in static cultures,

but, as expected from conditions close to microaerobiosis, values of both X (0.73, 1.35 and 1.38 g L-

1
in CW1, CW2 and CW3) and  (0.329, 0.385 and 0.402 h-1, respectively) were remarkably lower.

On the other hand, the yield of biomass on consumed lactose (YX/S) achieved its maximum value

(0.083 g g-1) on CW1 under agitation, the minimum value (0.027 g g-1) on the same whey permeate

in static culture, and an intermediate one (about 0.065 g g-1) on the other two media under either

condition (Fig. 1B). The results of YX/S as a whole suggest a complex combination of cheese whey

permeate concentration and agitation on biomass growth, i.e. a negative effect of the former likely

due to substrate saturation and/or excess osmotic pressure increase, and a positive one of the latter

probably associated to the rate of substrate diffusion from the bulk to the cell.

Figure 1

The above X values were lower than those reported for Kluyveromyces marxianus cultivated

in cheese whey permeate with So = 25.0-85.0 g L-1 under aerobic (3.6-9.2 g L-1) and anoxic (1.5-2.8

g L-1) conditions [38], while those of  were similar in the former case (0.429-0.450 h-1) and higher

in the latter (0.250-0.260 h-1). Consistently with the well-known Crabtree-negative phenotype of

Kluyveromyces spp., i.e., a predominantly oxidative metabolism, these results confirm the difficulty

of both species to grow under conditions approaching anaerobiosis [29].

9
3.2 Lactose consumption and ethanol production

One can see in Figure 2 that, since some aeration is needed to sustain the respirofermentative

metabolism of K. lactis [27,39], ethanol production was faster than in static culture, but its

maximum concentration was lower.

Figure 2

But, whereas in static cultures the use of progressively more concentrated CW led to an

almost generalized enhancement of ethanol production parameters (Table 1; Fig. 2B), the opposite

occurred in shake flasks likely due to the Crabtree-negative phenotype of this microorganism that

favored growth.

Table 1

Lactose consumption was almost complete after 48 h under mixing, almost irrespectively of

whey permeate concentration (Fig. 2A). On the other hand, static cultivations on CW1 and CW2

took no less than 72 h to almost completely uptake lactose, while only 82% of the initial lactose was

consumed on CW3 even after 96 h (Fig. 2B), which means that either an increase in concentration

or the air starvation due to lack of agitation affected lactose consumption.

As a typical growth-associated product, ethanol was mainly produced during the exponential

growth phase, and its highest concentrations were always observed in the early stationary phase,

i.e., after 48 h in shake cultures carried out on CW1 (15.0 g L-1) and CW2 (10.0 g L-1) or after 72 h

in the slower ones carried out without agitation on CW2 (21.0 g L-1) and CW3 (22.2 g L-1) (Fig. 2).

These results taken together are consistent with those reported for batch ethanol production by

another K. lactis strain from a different cheese whey, which started in the exponential growth phase

and achieved its maximum value in the stationary one [39].

10
 Figure 2 also illustrates the time behavior of the yield of ethanol on consumed lactose (YE/S)

in batch cultures carried out with and without agitation, respectively. Under agitation, this yield

achieved a maximum value (0.63 g g-1) after 24 h in CW1, while it progressively decreased with

concentrating cheese whey permeate (to around 0.2 and 0.1 g g-1 for CW2 and CW3, respectively)

and almost negligibly varied along the time (Fig. 2C). For static culture, YE/S varied little in the

different media, with values between 0.27 and 0.34 g g-1 (Fig. 2D). However, for CW2 and CW3 it

was higher than under agitation, which means that, under these conditions, an increase in lactose

concentration benefited the yield of ethanol, even though its value was still lower than that observed

in CW1 under agitation.

In shake flasks, significantly higher fermentative parameters of ethanol production were

obtained in CW1 compared with CW2 and CW3 (p < 0.05) (Table 1). Lower final ethanol

concentration (Ef ≈ 5.50 g L-1) and yield of ethanol on consumed lactose (YE/S = 0.30 g g-1) were

reported for batch cultures of K. lactis in shake flasks on CW permeate with approximately 89 g L-1

lactose [39] and on milk whey [40], respectively. On the other hand, in static cultures Ef (21.0-22.2 g

L-1), YE/S (0.34-0.38 g g-1) and fermentation efficiency (FE = 63.2-71.0%) were significantly higher

in concentrated media compared with CW as such (Table 1), while the highest specific ethanol

productivity (qE = 0.360.11 g g-1 h-1) occurred in this last medium.

In designing ethanol production from cheese whey, a compromise must be reached between

maximization of ethanol production and minimization of residual sugar concentration, since one of

the purposes of the process is often the treatment of the by-product [1,8]. With that in mind, a

comparison of results in Table 1 shows that the largest ethanol productions associated with the

highest lactose removals and volumetric productivities were observed in concentrated CWs in static

cultures.

A comparison with K. marxianus, which stands out in lactose conversion to ethanol, reveals

that its performance is highly variable and strongly depends on the type of whey. To provide only a

few examples, using cheese whey permeate to cultivate this yeast under aerobic conditions, at S0

11
values comparable with those of the present work, Silveira et al. [38] obtained ethanol

concentrations ranging from 1.84 to 23.3 g L-1, with YE/S, yield of ethanol on biomass (YE/X) and

ethanol volumetric productivity (QE) in the ranges 0.11-0.34 g g-1, 0.73-3.37 g g-1 and 0.07-0.74 g L-
1
h-1, respectively. Koushki et al. [12] observed that ethanol production by K. marxianus (46.0 g L-1)

in twice-concentrated cheese whey permeate was more than twice that obtained in whey permeate

as such (S0 = 49 g L-1), and that FE and lactose consumption percentage were > 90 and 96%,

respectively. Finally, a fermentation yield between 0.22 and 0.53 g g-1 was reported for whey

fermentations by different K. marxianus strains, sometimes approaching even the theoretical yield

(0.54 g g-1) [41].

3.3 β-Galactosidase activity

The specific β-galactosidase (β-gal) activity was determined for cells grown on cheese whey

permeate either with or without agitation (Fig. 3). Cells cultured in shake flasks on CW1 exhibited,

on average, the lowest enzyme specific activities detected in this study (449.3-680.0 U g-1) (Fig.

3A), with no statistically significant difference among them (p > 0.05). In CW2, this activity

reached the highest value (1,783.926.7 U g-1) in the early stationary growth phase (24 h) and then

fell to less than half. Finally, in CW3 there was no statistically significant difference between the

values observed in the exponential growth phase after 8 h (962.5127.7 U g-1) and the late

stationary one after 72 h (798.622.4 U g-1).

Figure 3

The specific β-gal activity of cells cultured statically on CW1 and CW2 achieved maximum

values of 2,508.387.2 U g-1 and 2,468.442.3 U g-1, respectively, in the exponential growth phase

(8 h), and then decreased to 1,231.8-1,510.3 U g-1 and 2,177.7124.6 U g-1 in the late stationary one

(Fig. 3B), while in CW3 there was no significant difference among the high values (2,083.0-2,594.4
12
U g-1) obtained after different cultivation times. These results suggest that β-gal activity may

depend not only on the ability of cells to grow but also on cell concentration, consistently with its

well-known intracellular localization in K. lactis.

A decline in K. lactis β-gal activity under conditions where lactose was still present in

significant amounts in the medium was reported earlier [42], while Ornelas et al. [39] observed that

it declined at low lactose concentration but reached a maximum value when this sugar was

completely consumed, and ethanol began to be consumed. On the other hand, the β-gal activity

never seemed to be influenced by ethanol level under any of the conditions tested in the present

study, but appeared to simultaneously depend, also, on agitation conditions and CW composition. In

fact, in shake flasks on CW1 and CW3, it seemed not to be influenced by lactose concentration,

while in CW2 a decline occurred at low lactose concentration after 48 h (Figs. 2A and Fig. 3A). In

static cultures, the β-gal activity of cells grown on CW1 and CW2 decreased when lactose

concentration was reduced to less than 50% of the initial one, while it kept unchanged in CW3

(Figs. 2B and 3B).

Comparing cells grown in shake flasks, those cultured on CW2 exhibited the statistically

highest (p < 0.05) specific β-gal activities (1,534-1,784 U g-1) in the exponential growth phase (8 h)

and the early stationary one (24 h) (labeled with the same number 1 in Fig. 3A). On the other hand,

those cultured without agitation achieved remarkably higher activities (2,100 U g-1) (labeled with

the same number 2 in Fig. 3B) in CW1 after 8 h, in CW2 after 8 and 72 h and in CW3 along the

whole cultivation. These results are consistent with those reported by other authors [39], who

detected two maximum peaks of specific β-gal activity during batch aerobic cultures of K. lactis in

cheese whey, one at the late exponential growth phase and the other at the stationary one.

Resuming, a protocol aiming to maximize K. lactis β-gal activity in cheese whey permeate

should adopt static culture in twice-concentrated medium (CW3), which was the only one allowing

for β-gal activity 2,100 U g-1 throughout the whole fermentation.

13
3.4 Bioethanol production versus β-galactosidase activity

A final attempt was made to check the existence of a possible relationship between β-gal activity

and ethanol production. In shake cultures, maximum ethanol concentration (Fig. 1A) and maximum

specific β-gal activity of K. lactis cells (Fig. 3A) were concomitant in CW1 and CW3, but not in

CW2, whereas without agitation the same took place in CW2 and CW3, but not in CW1 (Fig. 3B);

therefore, it could not be established any direct correlation between these two activities. As

mentioned in the previous section, this observation could have been the result of a complex

combination of at least four factors, the growth status of cells, cell concentration, agitation

conditions and CW composition.

In order to identify a possible relationship between specific β-gal activity and ethanol yield

on consumed lactose over the agitated and static cultures in differently concentrated whey

permeates, Eq. (3) was used to calculate the linear correlation coefficient (r) [37], whose values are

listed in Table 2. According to this author, r values in the ranges 0.10-0.29, 0.30-0.49 and 0.50-1.0

should be considered small, medium and large, respectively. It can be seen that the values of such a

coefficient were always negative, pointing out inverse correlation between the selected two

variables. Moreover, the correlation was strong for both static and agitated cultures in CW1, as well

as for the static one in CW2, but weak for the others. In general, the closer to -1 the r value, the

greater the degree of negative linear statistical dependence, while the closer to 0, the lower the

strength of correlation between variables. Based on these results, it is possible to conclude that there

was no similarity between the distributions of scores of β-gal activity and YE/S.

This conclusion agrees with that of de Figueroa et al. [43], who observed in synthetic

medium that, contrary to expectations, the rate of lactose fermentation to ethanol by K. lactis was

not related to that of lactose hydrolysis. Oppositely, Gupte and Nair [44], working on cheese whey

as such in shake flasks, observed maximum β-gal activity of K. marxianus cells coincident with

optimal ethanol production. Such a concurrency was also detected for K. marxianus under hypoxic

conditions [45].

14
4 Conclusions

The aim of this work was to find a possible solution to the high polluting power of cheese whey

permeate by fermenting it with Kluyveromyces lactis, producing at the same time bioethanol and

biomass rich in β-galactosidase activity. To this purpose, we tested either different fermentation

conditions (shake flasks or static cultures) or medium concentrations (whey permeate as such or

1.5-fold- or twice-concentrated permeate). Ethanol was the main fermentation product either in

shake flasks or static cultures, with the highest production occurring after the exponential growth

phase. The use of a twice-concentrated permeate in static culture was found to be the best condition

to maximize both ethanol concentration (22.2 g L-1 after 72 h) and β-gal activity (2,083.0-2,594.4 U

g-1 over the whole fermentation), whereas, as expected, the highest biomass concentration (3.25 g L-
1
) was obtained in the same permeate using shake flasks. However, no direct correlation between

maximum ethanol concentration and specific β-gal activity could be established. Aiming to

simultaneously treat whey permeate and maximize both K. lactis β-gal activity and ethanol

production, a compromise may be found fermenting twice-concentrated permeate in static cultures,

where β-gal activity, fermentation efficiency and lactose conversion were the highest. These

findings, suggesting whey permeate exploitation as feedstock for industrial applications, may be

used as starting basis for advance in this field.

Acknowledgements

The authors thank Brazilian CNPq for the post-doc fellowship of Dr. F.C. Sampaio.

Conflict of Interest The authors declare that they have no conflict of interest.

15
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Captions of figures

Figure 1. Batch fermentations of cheese whey by Kluyveromyces lactis. (A) Maximum specific

growth rate and (B) dry biomass concentration after 24 h in shake flask (white bars) or in static

culture (gray bars). (B) Yield of biomass on consumed lactose in () shake flask or () static

culture. Media: cheese whey permeate as such containing 38.0 g L-1 lactose (CW1), 1.5-fold

concentrated cheese whey permeate containing 55.0 g L-1 lactose (CW2), twice-concentrated cheese

whey permeate containing 75 g L-1 lactose (CW3). Values are means of three replicates. Those

followed by the same lowercase letter do not differ significantly by the Tukey’s test (p > 0.05)

Figure 2. Batch fermentations of cheese whey permeate by Kluyveromyces lactis. Lactose uptake

(dotted curve) and ethanol production (full curve) with (A) and without (B) agitation. Ethanol yield

on consumed lactose with (C) and without (D) agitation. Media: cheese whey permeate as such

containing 38.0 g L-1 lactose (CW1) (), 1.5-fold concentrated cheese whey permeate containing

55.0 g L-1 lactose (CW2) (), twice-concentrated cheese whey permeate containing 75.0 g L-1

lactose (CW3) (). Values are means of three replicates. Standard deviation never exceeded  8%;

therefore, it is not displayed

Figure 3. Specific β-galactosidase activity of Kluyveromyces lactis cells grown in batch cultures

with (a) and without (b) agitation on cheese whey permeate as such containing 38.0 g L-1 lactose

(CW1) (gray bar); 1.5-fold concentrated cheese whey permeate containing 55.0 g L-1 lactose (CW2)

(white bar); twice- concentrated cheese whey permeate containing 75.0 g L-1 lactose (CW3) (black

bar). Values are means of three replicates. Those labeled with the same lowercase letter along the

time for the same type of medium do not differ significantly by the Tukey’s test (p > 0.05). Means

labeled with a number (1 or 2) are the statistically highest ones under each agitation condition by

the Tukey’s test (p  0.05)

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Table 1. Fermentative parameters of batch ethanol production by Kluyveromyces lactis in cheese whey permeatea)

Shake flasks Static cultures

Ethanol YE/S YE/X QE qE FE Ethanol YE/S YE/X QE qE FE

Wheyb) (g L-1)c) (g g- (g g- (g L- (g g-1 (%)h) (g L-1)c) (g g- (g (g L- (g g-1 (%)h)

)
1 d)
)
1 e) 1
h- h-1)g) )
1 d)
g- 1
h- h-1)g)
1 f)
) )
1 e) 1 f)
)

CW1 15.0  0.62  8.60  0.21  0.13  87.9 10.7  0.27  15.2 0.22  0.36  50.3 

0.7aB 0.02aA 0.07aB 0.01aB 0.02aC  0.5bC 0.01bC  0.01bB 0.11aA 14.4bC

4.3aA 0.6aA

CW2 10.0  0.23  3.35  0.21  0.07  42.2 21.0  0.34  15.8 0.29  0.22  62.3 

0.5bC 0.01bD 0.18bC 0.01bB 0.00bD  1.0aA 0.02aB  0.01aA 0.01bB 2.9aB

2.8bD 0.8aA

CW3 6.88  0.09  2.13  0.09  0.03  18.1 22.2  0.34  16.4 0.31  0.23  62.4 

0.62cD 0.01cE 0.10cC 0.01cC 0.00cD  2.2aA 0.02aC  0.01aA 0.03bB 3.0aC

1.6cE 1.6aA

Values are means of three replicates and refer to the time of maximum ethanol production (Shake flasks: 48 h in CW1 and
a)

CW2, 72 h in CW3; Static cultures: 48 h in CW1, 72 h in CW2 and CW3). Those followed by the same lowercase letter in
the same column or by uppercase letter in the same parameter for aerobic and microaerobic growth do not differ significantly
by Tukey’s test (p0.05), b)CW1 = cheese whey permeate as such with starting lactose concentration (So) of 38.0 g L-1; CW2
and CW3 = concentrated cheese whey permeates with S0 = 55.0 and 75.0 g L-1, respectively, c)
Maximum ethanol
concentration, d)Ethanol yield on consumed lactose, e)Ethanol yield on biomass, f)Volumetric ethanol productivity, g)Specific
ethanol productivity, h)Fermentation efficiency.

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Table 2. Coefficient of linear correlation (r) determined between the specific β-gal activity (U g-1)

and ethanol yield on consumed lactose (g g-1) in different cheese whey permeates

Wheya Shake flasks Static cultures

CW1 -0.86 -0.90

CW2 -0.17 -0.63

CW3 -0.46 -0.43

CW1 = cheese whey permeate as such with starting lactose

a)

concentration (So) of 38.0 g L-1; CW2 and CW3 = concentrated cheese

whey permeates with S0 = 55.0 and 75.0 g L-1, respectively.

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