Catherine Teixeira de CARVALHOa, Sérgio Dantas de OLIVEIRA JÚNIORa, Wildson Bernardino de Brito LIMAa, Fábio Gonçalves Macêdo de MEDEIROSb, Ana Laura Oliveira
de Sá LEITÃO a, Everaldo Silvino dos SANTOSa, Gorete Ribeiro de MACÊDOa, Francisco Canindé de SOUSA JÚNIORc *
a Laboratory of Biochemical Engineering, Chemical Engineering Department, Federal
University of Rio Grande do Norte (UFRN), 59078-970, Natal-RN, Brazil
b Bioprocess Laboratory, Chemical Engineering Department, Federal University of Rio Grande
do Norte (UFRN), 59078-970, Natal-RN, Brazil
c Laboratory of Bromatology, Department of Pharmacy, Health Sciences Center, Federal
University of Rio Grande do Norte (UFRN), 59012-970, Natal-RN, Brazil
*Corresponding author:
Francisco Canindé de Sousa Júnior
Laboratory of Bromatology, Department of Pharmacy, Health Sciences Center, Federal University of Rio Grande do Norte (UFRN), 59012-970, Natal, Brazil. E-mail: fcfarma@yahoo.com.br
Abstract
The present study evaluated the co-production of β-galactosidase and ethanol by yeasts
Kluyveromyces marxianus ATCC 36907 and Kluyveromyces lactis NRRL Y-8279 using lactose
from "coalho" cheese whey as a carbon source. Cheese whey was subjected to partial deproteinization and physicochemical parameters were assessed. Submerged cultivations were carried out in an orbital shaker to evaluate two carbon/nitrogen (C:N) ratios, 1.5:1 and 2.5:1. The best C:N ratio (1.5:1) was carried to 1.5-L bioreactor cultivation in order to increase co- production yields. Stability of β-galactosidase was assessed against different temperatures and pH, and in the presence of metal ions. Concerning the co-production of β-galactosidase and ethanol, Kluyveromyces lactis NRRL Y-8279 proved to be more efficient in both the C:N ratios, reaching 21.09 U/mL of total enzyme and 7.10 g/L of ethanol in 16 h, for bioreactor cultivations. This study describes the development of a viable, value-adding biotechnological process using regional cheese by-product from northeast Brazil for co-production of biomolecules of industrial interest.
1. Introduction
The dairy industry plays an economically important role on the agri-food sector, and its annually growing production is directly related to the increasing generation of wastes and by- products, such as cheese whey (CW) (Escalante et al., 2018). The global CW generation is estimated at around 200 million tons per year, with a linear increasing tendency (Domingos et
al., 2018; Treu et al., 2019). In Northeast Brazil, the CW production is intrinsically related to
the “coalho” cheese industry. The “coalho” cheese is a highly traditional Brazilian cheese, which assumes relevant socio-economical and nutritional components in the Northeast region (Fontenele et al., 2017; Soares et al., 2017). The CW is a by-product with significant pollutant potential due to its organic matter content and high biological oxygen demand (BOD; Andrade
et al., 2017). On a dry basis, the composition of CW can reach up to 80% of lactose, one of the
most environmentally harmful sugars (Zhou et al., 2019).
Several strategies have been investigated for dealing with the CW waste disposal and the use of biotechnological processes figures as an interesting way of converting such by- product into a valuable feedstock (Carota et al., 2017). Although difficult to degrade on the environment, the lactose content of CW can be used as platform for the fermentation of value- added products such as ethanol (Beniwal et al., 2018), galactonic acid (Zhou et al., 2019), and β-galactosidase (Perini, et al., 2013; Rao e Dutta, 1977). The enzyme β-galactosidase (β-gal; EC 3.2.1.23), also known as lactase, is a product of great interest and several applications in the food industry, as it is responsible for the hydrolysis of lactose glycosidic bonds (Panesar et al., 2018a). In addition, to the increasing market share of lactose-free products for diet-restricted consumers (Suri et al., 2019), β-gal is also used for the enzymatic production of food prebiotics such as lactulose and different galactooligossacarídeos (GOS) ( Nooshkam et al., 2018; Panesar et al., 2018).
Yeasts from the genus Kluyveromyces spp. have been used for the production of β-gal enzyme from lactose-based substrates for generally recognized as safe (GRAS) applications for human consumption (González-Delgado et al., 2016; Perini, et al., 2013). Kluyveromyces spp. yeasts present a respiratory-fermentative metabolic pathway that can generate energy through the Krebs cycle (citric acid cycle) or by exclusive fermentation, in which ethanol is the main product. In contrast to Saccharomyces spp. genus, yeasts with respiratory-fermentative metabolism do not fully exploit their ability to uptake glucose during oxidative growth and, therefore, present a Crabtree effect. In fact, Kluyveromyces spp. yeasts are able to simultaneously perform fermentation and respiratory processes, and the balance between these two metabolic pathways depends on the specificity of the lineage (Lane e Morrissey, 2010).
In a recent study, You et al. (You, Chang, et al., 2017) proposed the utilization of whey powder as a substrate for low-cost production of β-gal enzyme. In addition, the authors used a co-production strategy in which the ethanol, a fermentation by-product, could also be recovered. Thus, the present study investigates the use of “coalho” cheese whey as a substrate for the low-cost co-production of β-gal enzyme and ethanol by Kluyveromyces marxianus and
Kluyveromyces lactis. To the best of our knowledge, this is the first report in the literature that
describes the use of this regional industrial by-product for co-production of these biomolecules thus showing an interesting biotechnological process.
2. Material and methods 2.1. Reagents
Yeast extract was acquired from Exôdo Científica (Brazil), dextrose, potassium phosphate and magnesium sulfate were acquired from Synth (Brazil), peptone was acquired from Kasvi (Brazil), agar was acquired from Vetec (Brazil) and ammonium sulphate was acquired from Cinética (Brazil).
2.2 Cheese whey (CW) obtaining and treatment
The cheese whey (CW) used in this study was obtained from a small "coalho" cheese producer in the city of Ceará Mirim (Rio Grande do Norte, Brazil) and kept at -20° C until use. For use in the process, the cheese whey partially deproteinized (CWD) was obtained by acid precipitation followed by thermal coagulation. For this process, CW (250 mL) was heated to 85 °C for 15 min and 3 mL of citric acid 10% was added. After that, the acidified mixture was heated to 90 °C for 15 minutes. The CWD was filtered with cheese cloth and kept at -20 °C until further use (Koushki et al., 2012; Florêncio et al., 2013).
2.3 Yeast strains and inoculum preparation
Kluyveromyces marxianus ATCC 36907, donated by the Federal University of Ceará
(UFC, Brazil), and Kluyveromyces lactis NRRL Y-8279, provided by the ARS Collection culture (Peoria, Illinois, USA) were used in this study.
Both yeasts were stored in 30% (v/v) glycerol at -20 °C. For the preparation of the inoculum, the microorganisms were cultured in YEPD medium (10 g/L yeast extract, 20 g/L dextrose, 20 g/L peptone e 20 g/L agar) for 24 h, at 30 °C. Three isolated colonies were transferred to 50 mL of the culture medium containing 10 g/L lactose, 5 g/L of potassium phosphate, 1.2 g/L of ammonium sulfate, 1.0 g/L yeast extract 0.4 g/L of magnesium sulfate prepared in potassium phosphate buffer (0.2 M, pH 5.5) according to Lima et al.(2013), and incubated for 16 h, at 180 rpm and 30 °C. Lactose used in the growth medium was exclusively from QWD.
2.4. Carbon/nitrogen ratio assessment
The co-production of β-galactosidase (β-gal) and ethanol was evaluated in submerged cultivation using orbital shaker and bioreactor. Two carbon/nitrogen ratios (C:N) were evaluated for the co-production process (1.5:1 and 2.5:1) according to the conditions described
by (You et al., 2017b). For all cultivations, lactose from CWD was used as the sole source of carbon available in the growth medium. Table 1 describes the complete compositions of culture media for the two C:N ratios in this study.
For each of the co-production experiment, the media pH, lactose concentration (g/L), ethanol concentration (g/L), total enzymatic activity of β-gal (U/mL), β-gal specific enzymatic activity (U/mg) and total protein (mg/mL) were evaluated.
Table 1 - Complete composition of the culture media used for the two carbon/nitrogen (C: N)
ratios ratio) in this study. Lactose from partially deproteinized cheese whey (CWD) was used as a sole source of carbon.
C: N ratioa Lactose (g/L) (NH4)2SO4 (g/L) Yeast extract (g/L) KH2PO4 (g/L) MgSO4.7H2O (g/L) 1.5:1 20.0 1.3 12.0 5.0 0.4 2.5:1 40.0 1.6 14.4 5.0 0.4
2.4.1 Culture conditions for the orbital shaker
Orbital shaker (model TE-241, Tecnal, Brazil) cultivations were conducted in 250 mL flasks with 50 mL growth medium, at 30 °C and 180 rpm. The inoculum was prepared as described in Section 2.2 and corresponded to 10% (v/v) of the final media volume. The culture medium was previously sterilized. CWD (lactose source) was filtered through a 0.22 μm membrane and added to the media under sterile conditions. Samples were taken after 4, 8, 12, 16, 20, 24, 36 and 48 h of fermentation
2.4.2. Culture conditions in the bioreactor
Bioreactor cultivations were conducted in a 2-L fermenter (model Biostat B, B-Braun, USA). The bioreactor was sterilized with 1.5-L of culture media and lactose from CWD was filtered through a 0.22 μm membrane and added moments before the start of the fermentation. The inoculum was prepared as described in Section 2.2 and corresponded to 10% (v/v) of the final media volume (Perini et al., 2013). The experiments were conducted in the following conditions: 30° C, pH 5.5 (controlled with solutions of 1.0 M HCl and 1.0 M NaOH), 200 rpm agitation and aeration of 1.33 vvm. Samples were taken after 4, 8, 12, 16, 20, 24, 36 and 48 h of fermentation.
2.4.3 Enzyme extraction
Due to the intracellular production of β-gal, the enzymatic extract was obtained from mechanical disruption of the yeasts cellular structure, as described by Braga et al.(2014), with minor modifications. The procedure was performed in 50 mL falcon tubes with 25 mL of cell suspension and 27.5 g of glass beads (diameter ranging from 0.95 to 1.05 mm), agitated in a vortex for 5 min, followed by 1 min of an ice bath. This procedure was repeated four times. The suspension was then centrifuged at 5200 ×g for 10 min, 4 °C (model CT-5000R, Solab, Brazil). The enzymatic extract was collected and stored at -20° C until use.
2.5 β-galactosidase stability assays
The β-gal stability was assayed at different pHs, in the presence of metal ions and at different temperatures based on the remaining activity after exposure, according to Oliveira et
al.(2018). After each treatment, the β-gal activity was determined as described in Section 2.4,
and the results were expressed as the enzymatic activity retention.
For pH stability, the enzymatic extract was exposed to pH 4.0 to 10.0, at 25 °C, for 30, 60 and 90 min. The following buffer solutions were used: sodium acetate (0.2 M, pH 4.0 – 5.0),
sodium phosphate (0.2 M, pH 6.0-8.0), glycine-NaOH (0.2 M, pH 9.0-10.0). For thermal stability, the enzymatic extract was exposed to temperatures of 40, 45 and 50 °C, at pH 6.6, for 15, 30 and 60 min. The effect of metal ions on the β-gal activity was assessed as described by Dias et al.(Dias et al., 2002), with minor modification. The enzymatic extract was exposed to solutions containing ions Mg2+ (MgSO4·7H2O, 5 mM), Cu2+ (CuSO4 ·5H2O, 5 mM),
Zn2+(ZnSO4 ·7H2O, 5 mM), Mn2+ (MnCl2, 5 mM), Fe2+ (FeSO4, 5 mM) e Co2+ (CoCl2, 5 mM),
at 25 °C, for 30, 60 and 120 min.
2.6 Analytical methods
The physicochemical parameters of CW and CWD samples were analyzed according to AOAC methods (AOAC, 2006). Briefly, total protein content was assessed by Kjeldahl method (method 991.20), ash content was analyzed by incineration at 550 °C (method 900.02), and pH was determined using a digital potentiometer (model pH 2600, Instrutherm, Brazil; method 981.12). Lipid content was determined by the Bligh & Dyer method (Gusso et al., 2012). The moisture content was determined by the gravimetric method at 105 °C (Bueno et al., 2017). The lactose content was evaluated by High-performance Liquid Chromatography (HPLC; model Ultimate 3000, Thermo Fisher Scientific, USA) using a Shim-Pack column (model SCR- 101H, Shimadzu, Japan) with refraction index detector (RID), at 40 °C. Sulfuric acid (0.005 M) was used as a mobile phase, under a flow of 1.0 mL/min. All samples were filtered through 0.22 µm membrane before analysis.
For all the cultivations, the biomass monitoring was carried through optical density (Spectrophotometer - model Genesys 10, Thermo Spectronic, USA), with reading at 620 nm (OD620; Lima et al., 2013) using an external calibration, and the results were expressed in g/L.
The concentrations of ethanol, lactose, glucose, and galactose were evaluated by HPLC, as described previously.
The enzymatic activity of β-gal was determined using the synthetic substrate O- nitrophenyl-β-D-galactopyranoside (ONPG), as described by Braga et al.(2014). The enzymatic activity of β-gal was expressed in U/mL. The enzymatic activity unit (U) was defined as the enzyme necessary to produce 1.0 μmol of O-nitrophenol per min at the assay’s conditions (37 °C, pH 6.6). The total protein was assessed by the Bradford method (Bradford, 1976). Bovine Serum Albumin (BSA) was used as standard and reads were performed at 595 nm. The protein concentration was expressed in mg/mL and used to calculate the specific activity of the enzyme β-gal (U/mg).
2.7 Kinetic parameters
The maximum specific growth velocity (𝜇𝑚𝑎𝑥), the maximum productivity of cells (𝑃𝑋) and products (𝑃𝑝𝑚𝑎𝑥; either 𝑃𝛽−𝐺𝑎𝑙 or 𝑃𝑒𝑡ℎ𝑎𝑛𝑜𝑙), and yield of cells (𝑌𝑋 𝑆⁄ )and products (𝑌𝑃 𝑆⁄ ;
either 𝑌𝛽−𝑔𝑎𝑙 𝑆⁄ or 𝑌𝑒𝑡ℎ𝑎𝑛𝑜𝑙 𝑆⁄ ) based on substrate (lactose) consumption were calculated using
Equations 1 – 5 (Schmidell et al., 2001; Vasconcelos et al., 2018).
ln𝑋𝑚𝑎𝑥 𝑋0 = 𝜇𝑚𝑎𝑥∙ (𝑡𝑓− 𝑡0) (1) 𝑃𝑋 = 𝑋𝑚𝑎𝑥−𝑋0 𝑡𝑓 (2) 𝑃𝑝𝑚𝑎𝑥 = 𝑃𝑚𝑎𝑥−𝑃0 𝑡𝑓 (3) 𝑌𝑋 𝑆⁄ =𝑋𝑆𝑚𝑎𝑥−𝑋0 0−𝑆𝑚𝑎𝑥 (4) 𝑌𝑃 𝑆⁄ = 𝑃𝑚𝑎𝑥−𝑃0 𝑆0−𝑆𝑚𝑎𝑥 (5)
Where 𝑋𝑚𝑎𝑥 is the maximum cells (biomass) concentration (g/L), 𝑋0 is the initial cell concentration (g/L), 𝑡𝑓 is the fermentation time to reach 𝑋𝑚𝑎𝑥 (h), 𝑆0 is the initial lactose
concentration (g/L), 𝑆𝑚𝑎𝑥 is the lactose concentration found when 𝑋𝑚𝑎𝑥 was achieved (g/L),
and 𝑃𝑝𝑚𝑎𝑥 is the maximum concentration of the products, either β-gal (𝑃𝛽−𝑔𝑎𝑙) or ethanol (𝑃𝑒𝑡ℎ𝑎𝑛𝑜𝑙).
From the maximum specific growth (𝜇𝑚𝑎𝑥), the generation time (GT) was calculated
with the following equation:
𝑡𝑔 = 𝑙𝑛𝑥
µ𝑚𝑎𝑥 (6)
2.8 Statistical analysis
All experiments were performed as three independent replicates (n = 3). Statistical analysis was performed using the software Statistica v. 8.0 (TIBCO Statistica, Palo Alto, CA, USA). One-way ANOVA combined with Tukey HSD post hoc test was applied to establish statistical significance (p < 0.05).
3. Results and Discussion
3.1 Physicochemical characterization of cheese whey
The results for the physical-chemical parameters of the cheese whey (CW) and cheese whey partially deproteinized (CWD) are presented in Table 2.
The differences in the composition of CW and CWD can be related to the partial deproteinization process, which altered the moisture and lactose contents, in addition to the protein composition of CW (p < 0.05). According to the pH, the CW used here is featured as "sweet whey" in agreement to Brazilian regulations (Brasil, 2013), but the acid treatment of the deproteinization lowed the pH of CWD to around 4.6. The partial deproteinization aims to
reduce the influence of the protein content of CW during the cultivations, besides, to increase the lactose concentration (Koushki. et al, 2012).
Table 2: Physicochemical parameters for cheese whey (CW) and cheese whey partially
deproteinized (CWD). Parameter CW CWD pH 6.81 ± 0.50a 4.60 ± 0.50b Ash (%) 6.91± 0.04a 6.46 ± 0.06a Protein (%) 10.39 ± 0.02a 5.40 ± 0.02b Fat (%) 2.31 ± 0.41a 2.16 ±0.33a Moisture (%) 94.71 ± 0.06b 97.17 ± 0.06a Lactose content (%) 2.70 ± 0.01b 4.40± 0.01a
Mean values ± Standard deviation. Different letters in the same line indicate statistical difference (p < 0.05) according to Tukey’s test.
The cheese whey is a cloudy liquid, yellow-green, which carries approximately 55% of the composition of the solids found in the whole milk. Lactose is the principal sugar found in the CW and is related to its high chemical oxygen demand (COD; Alves et al., 2014). Jacinto et al. (2012) evaluated the physicochemical composition of the CW used in the production of a fermented dairy drink and found similar results to the presented here, for moisture (92.13%), carbohydrates (lactose, 5.54%) and pH (6.46)
3.2 Co-production of β-galactosidase and ethanol in the orbital shaker
The co-production of β-gal and ethanol in the orbital shaker was investigated using two C:N ratios. Lactose from the CWD was the single carbon source used for all cultivations. K.
lactis NRRL Y-8279 and K. marxianus ATCC 36907 were used for the co-production using
C:N ratios of 1.5:1 and 2.5:1. The biomass and substrate (lactose) concentration profiles during the cultivations are presented in Fig. 1. Fig. 2 shows the co-production profiles of the two products, β-gal and ethanol. The kinetic parameters for all cultivations are presented in Table 3. The pH from the culture media during all the orbital shaker cultivations ranged from 4.95 to 5.47.
Fig. 1. Biomass growth profile for K. marxianus ATCC 36907 (●) and K. lactis NRRLY-8279
(○), and lactose consumption profile for K. marxianus ATCC 36907 (▲) and K. lactis NRRLY- 8279 (△) in the orbital shaker cultivations. (A) C/N 1,5:1; (B) C/N 2,5:1. Mean values ± Standard deviation.
Fig. 2. Co-production profile of β-galactosidase for K. marxianus ATCC 36907 (■) and K.
lactis NRRL Y-8279 (□) and of ethanol for K. marxianus ATCC 36907 (▼) and K. lactis
NRRLY-8279 (▽) in the orbital shaker cultivations. (A) C:N 1,5:1; (B) C/N 2,5:1. Mean values ± Standard deviation
Table 3: Kinetic parameters for the orbital shaker and bioreactor cultivations for the co-production of ethanol and β-galactosidase using cheese
aC: N: Carbon/nitrogen ratio.
bt: Cultivation time for the best co-production condition.
cX
a: Maximum cell concentration at the best co-production condition. dS
f: Final concentration of substrate (lactose). eS
0: Initial concentration of substrate (lactose). fμ
max: Maximum specific growth velocity gP
x, Pβ-gal, and Pethanol: Maximum productivity of cells, β-gal and ethanol
The biomass growth for both strains and lactose consumption profiles (Fig. 1A-B) presented a similar pattern, suggesting good metabolic adaptation to the culture medium based on CWD. The growth profile also suggested a high rate of cell growth, with generation time (GT) ranging from 4.62 h to 4.95 h, for the C:N ratios of 1.5:1 and 2.5:1, respectively, for K.
lactis NRRL Y-8279. The generation time presented by the K. marxianus ATCC 36907 for both
C:N ratios was similar (4.95 h), as shown in Table 3. In addition, the increase in the carbon concentration (C:N ratio from 1.5:1 to 2.5:1) resulted in a decrease of ethanol and β-gal in all cultivations. You et al. (2017a) conducted a similar study for the co-production of β-gal and ethanol from commercial whey powder and observed a similar relationship between the C:N ratio and the co-production yields, i.e., the increase of C:N ratio reduced the co-production of β-gal and ethanol. Also, Sampaio et al. (2019) recently conducted a similar study for the bioethanol production from cheese whey permeate and observed that ethanol production was increased after the yeast exponential growth.
The maximum specific growth velocity (μmax) ranged from 0.12 to 0.15 h-1 for both
strains in the orbital shaker cultivations. Sampaio et al. (2019) also investigated the co- production of ethanol and β-gal from cheese whey, using initial concentrations of lactose of 55 and 75 g/L in the culture medium and observed μmax between 0.46 and 0.48 h-1. On the other
hand, those authors observed low substrate conversion factors between 0.083g.g and 0.027g.g
1, which can be related to the high initial concentration of lactose in the culture media, due to
substrate inhibition.
For the K. marxianus ATCC 36907 cultivations, no significant difference (p > 0.05) was observed for the biomass concentrations between the two C:N ratios studied (14.38 ± 0.16 g/L and 13.89 ± 0.05 g/L, for C: N 1.5:1 and 2.5:1, respectively). However, for the C:N ratio of 1.5:1, the maximum co-production of β-gal and ethanol was observed after 16 h of cultivation, which accounted for a higher productivity factor (Px; 0.76 ± 0.01 g/L.h). Nonetheless, the overall production yields of β-gal for the K. marxianus ATCC 36907 cultivations were relatively low (Fig. 2A-B), reaching 92.49 and 66.23 U/g cells to the C:N ratios of 1.5:1 and 2.5:1, respectively. In addition, the yield of cells based on substrate (lactose) consumption (Yx/s)
for the K. marxianus ATCC 36907 cultivations ranged from 0.42 ± 0.01 to 0.99 ± 0.30 g.g-1, which are superior to the results found by Manera et al. (2008) in a study for the production of β-gal with K. marxianus using synthetic medium.
For the K. lactis NRRL Y-8279 cultivations, the maximum co-production of β-gal and ethanol were achieved after 20 h and 16 h of cultivation, for the C:N ratios of 1.5:1 and 2.5:1, respectively. After these peak productions, a decline in ethanol and β-gal production is observed. This behavior may be related to the batch strategy adopted, which did not involve further lactose retro-feeding strategy. The co-production yields for both products were significant different (p < 0.05) in the two C:N ratios. The lower C:N ratio (1.5:1) favored the production of β-gal, yielding 18.5 ± 0.01 U/mL of total enzyme concentration and 2340.08 U/g cells, for 18.58 g/L of lactose consumption and 9.01 ± 0.23 g/L of ethanol production, while the higher C:N ratio (2.5:1) favored the ethanol production (9.70 ± 0.29 g/L), for 38.98 g/L of lactose consumption and 770.10 U/g cells and 8.91 ± 0.38 U/mL.
You et al. (2017a) reported the best C:N ratio for the co-production of β-gal and ethanol of 2.5:1, using an initial concentration of 55 g/L of lactose from commercial whey powder. Additionally, they obtained 37.43 ± 2.6 U/mL of total β-gal activity and 21.42 ± 0.15 g/L of ethanol concentration. Although such results are higher than those obtained in the present study, it is important to note that the use of CWD takes into consideration the composition of an industrial waste as a source of nutrients, which may have been responsible for the differences in the results. In addition, the results of co-production presented here are quite promising since it proposes the use of cheese whey from small and mid-sized companies, which usually drain this kind of waste into the environment, for an innovative, viable biotechnological application, as a treatment for agro-industrial wastes (Silva et al., 2018).
From the results presented in Table 3, the best co-production of β-gal and ethanol condition was achieved for the cultivation in which the lowest biomass concentration was reached. Therefore, the bioreactor cultivations were carried using the yeast K. lactis NRRL Y- 8270 in the C:N ratio of 1.5:1.
3.3 Co-production of β-galactosidase and ethanol in bioreactor using K. lactis NRRL Y-8270
In order to increase the co-production yields of β-galactosidase and ethanol, the culture medium with C:N ratio of 1.5:1, with 20 g/L of lactose from the CWD, was used for the cultivation in a bioreactor with useful volume of 1.5 L. Biomass (cells) concentration and substrate consumption profiles are shown in Fig. 3. The kinetic parameters for the cultivations in bioreactor were calculated and are presented in Table 3. The pH of the culture medium in the bioreactor cultivations was controlled at 5.5, with no significant difference from the cultivations carried in the orbital shaker (from 4.95 to 5.5). According to Parazzi et al. (2017), this pH range favors the reproduction of yeasts and inhibits the growth of contaminating bacteria.
Fig. 3. (A) Biomass (cells) growth (●) and substrate (lactose) consumption (▲) profiles; and
(B) Co-production of β-galactosidase (○) and ethanol (□) for the bioreactor cultivations using
K. lactis NRRLY-8279 and the C:N ratio of 1.5:1. Mean values ± Standard deviation. .
The profile of cellular growth of the K. lactis NRRLY-8279 strain in the bioreactor cultivations was similar to the observed for the orbital shaker cultivations for the same processing time (48 h). The maximum co-production time in the bioreactor was 16 h (Fig. 3A),