Animal Science

Selection of a culture medium for the growth of Pichia guilliermondii LEVICA- 27 as activator additive of ruminal fermentation

0000-0001-6408-2859Bexy González Mora^{1  2  *}  , 0000-0003-3933-1176Dailyn Sosa Cossio¹  , 0000-0001-6213-5857Yoandra Marrero Rodríguez¹  , 0000-0002-7055-4880Yaneisy García Hernández¹  , 0000-0003-4827-3991Nereyda Albelo Dorta¹ 

¹Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas. Mayabeque, Cuba

²Facultad de Zootecnia y Ecología, Universidad Autónoma de Chihuahua (UACH), Perif. Francisco. R. Almada km 1, Zootecnia, Chihuahua, CP 33820. Chihuahua, México

ABSTRACT

In order to select a non-conventional culture medium for the growth of Pichia guilliermondii LEVICA-27 as an activator additive for ruminal fermentation, two trials were carried out in which YPG medium was used (yeast extract 10 g/L, peptone 10 g/L and glucose 20 g/L) as control medium. In the first, seven culture media were evaluated for yeast growth at 24 hours of fermentation. While in the second, growth kinetics was performed in the selected medium of the first test. In addition, in the latter, the maximum specific growth rate, the biomass doubling time, and the stoichiometric balance of the selected medium were determined. At 24 hours of fermentation, there were not differences between the media studied and the YPG medium (p=0.073). Therefore, the latter can be replaced without affecting the strain growth in 24 h. In the kinetic study, there was a higher microbial concentration at 12 h (P<0.0001) in the medium containing molasses and urea. The maximum specific growth rate was lower (P=0.0260) and the biomass doubling time was higher (P=0.0283) with respect to the control. The selected medium constitutes an adequate option for the production of LEVICA-27, since it allows obtaining good cell concentration, covering the nutritional requirements and includes fewer components in its preparation. This offers economic and operational advantages for obtaining the additive at production scales.

Key words: molasses; yeast; fermentation; biomass

The use of yeast additives in ruminant feeding constitutes an alternative to increase milk and meat production in the world (^{Elghandour et al. 2020} and ^{Suntara et al. 2021}). To obtain these additives through fermentation processes, it is important to select a culture medium that is economically feasible and covers the nutritional requirements of the yeast strain used.

It is estimated that approximately 30 % of the total cost of fermentation is the cost of the culture medium (^{Bharti et al. 2018}). The most used commercial media for yeast growth in laboratory studies are malt extract and YPG medium (yeast extract, peptone and glucose). However, the high prices they have in the market prevent their use at productive scales. For this reason, it is necessary to allocate efforts in the search for economic sources of nutrients for large-scale yeast production.

The use of agro-industrial by-products as substrates for microbial growth are widely studied because it provides carbon and nitrogen sources that is less expensive. In addition, it allows the use of wastes that should be treated before its disposal, and thus add value to the latter. Among the most used agro-industrial by-products are sugar cane molasses (^{Álvarez-Cao et al. 2019} and ^{Bento et al. 2020}) and whey (^{Cortez et al. 2019} and ^{Santiesteban-Lopez et al. 2020}). However, the results with the use of these unconventional media vary depending on the yeast strain used (^{Marrero et al. 2016} and ^{Marrero et al. 2020a}). For the above reasons, it is necessary to find the right components in the culture medium, so that they guarantee the highest possible growth.

The Instituto de Ciencia Animal (ICA) of the Republic of Cuba has a collection of yeasts isolated from the ruminal ecosystem of cows that previously intake a fermented product, rich in yeast (^{Sosa et al. 2018}). Among them is Pichia guilliermondii LEVICA-27, which in previous studies showed potential to stimulate ruminal fermentation in animals that intake fibrous diets (^{Marrero et al. 2020a} and ^{Marrero et al. 2020b}). The results justify the search for an adequate and economically feasible culture medium to obtain an additive with Pichia guilliermondii LEVICA-27 on an industrial scale and its subsequent introduction into livestock systems. Hence, the objective of this research was to select a non-conventional culture medium for the growth of Pichia guilliermondii LEVICA-27 strain.

Materials and Methods

The studies were carried out in Laboratorio de Producción de Alimentos de la Unidad Central de Laboratorios (UCELAB) belonging to Instituto de Ciencia Animal (ICA), located at Carretera Central km 47 ½, in San José de las Lajas, Mayabeque, Cuba.

Design and experimental treatments. Two tests were carried out to evaluate the growth of Pichia guilliermondii LEVICA-27 in culture media, from cheap substrates. In the first, a completely random design was used to compare the yeast growth in each medium to be evaluated (M2, M3, M4, M5, M6 and M7) with respect to the control medium YPG (yeast extract 10 g/L, peptone 10 g/L and glucose 20 g/L). Previously, the effect of different components that are usually reported for yeast growth was consulted. The M2 medium was proposed by ^{Sánchez et al. (2007)} for a strain of Pichia guilliermondii, destined for animal feeding. Then, modifications were made to this culture medium that consisted of the incorporation of some mineral sources. Table 1 shows the composition of the seven culture media evaluated in the first test.

Table 1 Composition of the culture media (M1, M2, M3, M4, M5, M6 and M7), evaluated for the growth of Pichia guilliermondii LEVICA-27 

Nutrient concentration (g/L)	(Control)
	M1	M2	M3	M4	M5	M6	M7
Sugar cane molasses	-	20	20	20	20	20	20
Yeast extract	10	-	-	-	-	-	-
Peptone	10	-	-	-	-	-	-
Glucose	20	-	-	-	-	-	-
Urea	-	10	10	10	10	10	10
KH2PO4	-	-	5	5	5	5	5
MgSO4	-	-	1.5	1.5	1.5	1.5	-
FeCl3·6H2O	-	-	0.15	0.15	0.15	-	-
CuSO4	-	-	0.1	0.1	-	-	-
MnSO4·H2O	-	-	0.06	-	-	-	-

Once the most suitable culture medium was selected from the technical and economic point of view for the culture of Pichia guilliermondii LEVICA-27, the second test was carried out. In this, a completely random design with a 2x10 factorial arrangement was used, in which the yeast growth in the previously selected medium was monitored. The factors were the culture medium and time (0, 2, 3, 4, 8, 12, 16, 20, 24 and 28 h). The YPG medium was used as control medium and three repetitions were performed at each sampling time for the two culture media studied.

Experimental procedure. The strain Pichia guilliermondii LEVICA-27, belonging to Banco de Microorganismos para la Producción Animal (BAMIPA) from the Instituto de Ciencia Animal (Mayabeque, Cuba), was used. The strain was activated by means of two subcultures in YPG medium at 110 r.p.m, 30 ºC and 24 h of incubation. From the active culture, the inoculum was obtained under the same conditions. It was inoculated at 10 % (v/v) in erlenmeyer flasks with a capacity of 100 mL, with 45 mL of the culture media corresponding to the experimental treatments. The initial concentration of Pichia guilliermondii LEVICA-27 in all the studied media was 10⁶ cfu/mL. The flasks were incubated in an orbital shaker at 30 ºC and 110 r.p.m. In the first test, sampling was done at 24 h. At this time, the flasks were shaken and 1 mL of the homogeneous sample was taken. All samples were serially diluted with saline (0.85 %, w/v) as a diluent and seeded in Petri dishes with Sabouraud agar (Biolife, Italy) to determine, by visual counting, the colony-forming units per milliliter (cfu/mL). In the second test, the same procedure as in the previous one was used to determine the cfu/mL at each sampling time. From this last indicator, the maximum specific growth rate (µ) of the strain was determined by linear regression of the ln (cfu/mL) in the exponential phase. In addition, the biomass doubling time (d_t) was calculated, according to equation 1.

${\mathrm{d}}_{\mathrm{t}}=\frac{\ln 2}{\mathrm{\mu }}\mathrm{}\mathrm{}\mathrm{}\mathrm{}$ (1)

The stoichiometric balance of the medium selected in the first study was performed. The necessary amount of carbon source in the culture medium was determined by the biomass/substrate yield (Y_X⁄_S), represented in equation 2. While, the rest of the components were determined by mass balance (equation 3), assuming that all the added mass of each element is transformed into a cellular component. The average elemental composition of a microorganism (CH_1.79O₀.₅₀N₀.₂₀) reported by ^{Doran (2013)} was used, with a standard deviation of 3 %. The microbial concentration in g/L was determined from a model fitted by linear regression that relates the dry weight of the biomass of LEVICA-27 with the cfu/mL.

${\mathrm{Y}}_{\raisebox{1ex}{$\mathrm{X}$}\!\left/ \!\raisebox{-1ex}{$\mathrm{S}$}\right.}=\frac{{\mathrm{X}}_{\mathrm{f}}-{\mathrm{X}}_0}{{\mathrm{S}}_0-{\mathrm{S}}_{\mathrm{f}}}\mathrm{}\mathrm{}\mathrm{}\mathrm{}\mathrm{}\mathrm{}\mathrm{}\mathrm{}$

Where:

X0 and Xf

are the initial and final concentrations of biomass (g/L) and S0 and Sf are the initial and final concentrations of substrate, respectively, g/L

${\mathrm{m}}_{\mathrm{i}}^0={\mathrm{w}}_{\mathrm{i}}\bullet {\mathrm{m}}_{\mathrm{x}}$

Where:

${\begin{array}{c} \\ m\end{array}}_{\mathrm{i}}^0$

is the initial mass of element i in the culture medium (g/L), wi is the mass fraction of element i in the cell and mx is the cell mass, g/L.

Statistical analysis. The data was processed with the statistical program Infostat (^{Di Rienzo et al. 2017}). In the first test, means were compared using orthogonal contrast (M1 vs. M2, M3, M4, M5, M6 and M7) by Scheffé's test (^{Montgomery 2004}). In the second, the comparison of means between treatments was performed using ^{Duncan (1955)} test.

Results and Discussion

Figure 1 shows the cell concentrations (log cfu/mL) of Pichia guilliermondii LEVICA-27 at 24 h of fermentation in the culture media evaluated. There were no differences between the studied media, when each one is compared with the control (M1). It was verified that with the culture media from unconventional sources concentrations of Pichia guilliermondii LEVICA-27 were obtained similar to those of the conventional medium used as control. Hence, the latter can be replaced by the others, without affecting the strain growth.

^{a, b} Different letters differ at P<0.05, SE ± 0.06, P=0.073

Figure 1 Concentration of P. guilliermondii LEVICA-27 in media M1 (control), M2, M3, M4, M5, M6 and M7 cultured at 30 ºC and 110 r.p.m, at 24 h 

It is important to highlight that the M2 medium, in addition to allowing the growth of LEVICA-27, in concentrations similar to the control, has the least amount of components. Its simplicity allows it to be the most suitable from a technical-economic point of view. This advantage facilitates the production of yeast at higher scales, in which the use of conventional media or with a large number of components is not feasible. Given the absence of statistical difference in the evaluated media with respect to the conventional medium used as control, the M2 medium was selected to be used in the growth of LEVICA-27. The reasons for its choice are related to its simplicity, in terms of components and low cost, since it only contains molasses and urea, which are economical sources of nutrients for yeast growth.

Most of the studies consulted in the scientific literature used a strain of Pichia guilliermondii for purposes different than animal feeding. In this study, different culture media were evaluated for the growth of Pichia guilliermondii LEVICA-27. ^{Roepcke et al. (2011)} and ^{Wang et al. (2012)} used culture media for the growth of Pichia guilliermondii, similar to M3, M4, M5 and M6, and found high concentrations of cell biomass (10 g/L). According to these results, it was expected to obtain a higher concentration of biomass in the media M3, M4, M5 and M6. However, there were no differences between the growth obtained with the control medium (M1) and the rest of the evaluated media, despite that they contained mineral sources that can stimulate cell growth. This unexpected result could be related to other variables of the fermentation process, such as the concentration of dissolved oxygen in the culture medium, the speed of agitation, temperature and pH. It is important to study in future researches these variables that also influence on yeast growth.

Molasses is a carbon source par excellence for the growth of microorganisms. In addition, it has minerals such as calcium, magnesium, iron, potassium, zinc and other growth factors (niacin, riboflavin, pantothenic acid) in its composition, which also favor cell growth, and could be the reason why LEVICA-27 it did not require other sources of minerals. ^{Do et al. (2019)} recognize that yeasts need vitamins and minerals for their development. However, they pointed out that the requirements can vary from one strain to another, so that there is no homogeneous behavior, in terms of the requirements of minerals or growth factors in yeasts. In the case of LEVICA-27, the sugar cane molasses, in addition to satisfying the carbon needs, provided the necessary minerals to obtain a growth similar to that obtained with the YPG commercial medium. The possibility of dispensing with these components makes it possible to simplify the culture medium, obtaining an acceptable biomass production without nutrient limitations.

In this test, urea was used, as it is the cheapest nitrogen source worldwide and its use covered the nitrogen needs, without affecting the growth of LEVICA-27. According to these results, ^{Marrero et al. (2016)}, when studying different nitrogen sources for the growth of this strain, found that the highest optical density values were observed with the use of casein and urea, a result that doubles that obtained with ammonium sulfate.

Figure 2 shows the growth kinetics of Pichia guilliermondii LEVICA-27 in the M2 and control (M1) media. There was no presence of the adaptation phase in any of the two culture media evaluated. In both cases, the duration of the exponential phase was approximately 12 h, when the maximum microbial concentration was obtained, with a value of 7.1 log cfu/mL in the M1 medium and 7.62 log cfu/mL in the M2 medium. The stationary phase extended from 12 to 28 h in the two culture media.

^{a,b,c,d,e,f,g,h,i} Different letters differ at P<0.05, SE ± 0.1, P<0.0001

Figure 2 Comparison of the growth kinetics of Pichia guilliermondii LEVICA-27 in media M1 (YPG medium) and M2 (molasses and urea) at 30 ºC, during 28 h of fermentation 

The absence of the adaptation phase showed in this test is a desired characteristic, especially in fermentations at the industrial level, because it allows reducing the production times. In agreement with this result, ^{Wang et al. (2012)} did not observe the presence of this phase, when they cultivated a strain of Pichia guilliermondi in a different medium from the one used for the inoculum in a laboratory bioreactor.

Unlike the previous test, LEVICA-27 had a higher concentration in the medium with unconventional sources than in the control medium. The strain reached its maximum growth in the M2 medium at 12 h (7.62 log cfu/mL), so in subsequent studies it will be convenient to stop the fermentation process at this time, where the maximum growth of LEVICA-27 is obtained.

Table 2 shows the results of the growth characteristics of LEVICA-27 strain in the last two studied media. The maximum specific growth rate was lower in the M2 medium compared to the control and, consequently, the biomass doubling time was higher.

Table 2 Growth characteristics of the Pichia guilliermondii LEVICA-27 strain in M1 (YPG) and M2 (molasses and urea) media 

Indicators	M1	M2	± SE Sign
Duration of the exponential phase, h	0-12	0-12	-
Maximum specific growth rate, h^-1	0.55 ± 0.07	0.36 ± 0.06	0.04 P = 0.0260
Biomass doubling time, h	1.28 ± 0.17	1.94 ± 0.29	0.14 P = 0.0283
Log Xmax (Xmax x 10^7 ufc/mL)	7.12 ± 0.11 (1.35)	7.62 ± 0.27 (4.80)	0.12 P = 0.0392

X _max is the maximum biomass concentration

Similar results were found by ^{Aguilar et al. (2015)} with Saccharomyces cerevisiae, cultivated in a medium with molasses and whey (7.81 log cfu/mL), approximately at 16 h of fermentation. These authors obtained 3.26 h as biomass doubling time and estimated the maximum specific growth rate using the modified Gompertz model and reported lower values (0.09 h^-1) than those of this research.

^{Sanchez et al. (2007)} reported values of maximum specific speed and biomass doubling time similar to those of this study, when evaluating the fermentation kinetics of Pichia guilliermondii strain with the same M2. However, the mentioned authors observed the presence of the adaptation phase lasting 4 h and the maximum concentration of biomass (3.97 g/L) was obtained at 24 h. This shows the specificity of microorganisms, since their behavior not only depends on the culture medium, genus or species, but also on the strain used.

In the stoichiometric balance, it was determined that the culture medium M2 must contain 2.32 g/L of total reducing sugars (3.87 g/L of sugar cane molasses) and 0.39 g/L of urea to produce 7.62 log cfu/mL (1.66 g dry biomass/L) which corresponds to the highest concentration of LEVICA-27 reached in this study. The balance also showed that the M2 medium, composed of 20 g/L of sugar cane molasses with 12 g/L of total reducing sugars (60 %) and 10 g/L of urea, has the potential to obtain 4 g dry biomass/L (10⁹ cfu/mL) of LEVICA-27, so there is no nutrient limitation.

Regarding the economic analysis of these two media, molasses and urea have a market cost of 2.4 and 2 USD/kg, respectively. On the other hand, the kilogram of YPG medium has a cost of approximately 260 USD. If the concentrations of the components in each culture medium are considered, it is obtained that the production of 100 L of YPG medium has a cost of 400 USD, and this same amount of medium M2 (molasses and urea) will have a cost of 6.82 USD. Hence the cost of the latter represents only 1.7 % of the conventional medium. In addition to the economic aspect, the use of simple culture media is very important for biomass production at industrial levels. This is due to the reduction of the time that is used in its preparation during the technological process.

When analyzing all the indicators, it can be stated that the M2 medium is the most suitable option for the production of Pichia guilliermondii LEVICA-27 as an additive for animal feeding. In this case, in addition to obtaining better growth parameters than in the control medium, it includes fewer components in its preparation and has a lower cost, which offers economic and operational advantages for obtaining the additive at productive scales.

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Received: February 04, 2022; Accepted: March 28, 2022

^*Email: [e-mail], [e-mail]

Conflict of interests: The authors declare that there is no conflict of interest between them.

Authors contribution: Bexy González Mora: Conceptualization, Investigation, Writing - original draft. Dailyn Sosa Cossio: Investigation, Supervision, Data curation, Formal analysis. Yoandra Marrero Rodríguez: Data curation, Formal analysis, Writing - original draft. Yaneisy García Hernández: Supervision, Data curation, Writing - original draft. Nereyda Albelo Dorta: Investigation, Data curation, Formal analysis.