Influence of carbon source , agitation and aeration rates for production yeast biomass which potential of use for biological control

1 Influência da fonte de carbono, taxas de agitação e aeração na produção de biomassa de levedura com potencial de uso no controle biológico Influence of carbon source, agitation and aeration rates for production yeast biomass which potential of use for biological control Influencia de la fuente de carbono, las tasas de agitación y aireación en la producción de biomasa de levadura con potencial para su uso en el control biológico


Introduction
The international market has been attentive to quality and safety of agricultural products. Consumers and producers are both concerned that agricultural products are free from chemical residues. Other issues are the high cost of synthetic pesticides and the regular use of these components which contributes to the appearance of resistant strains of fungi (Gouvea, 2007). Development, v. 9, n. 4, e174943066, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i4.3066 3 In this context, biological control is a viable alternative to control pests and pathogens and also advantageous compared to chemical control, particularly with regard to environmental impact, cost, specificity and resistance development (Silveira et al., 2005;Schrank et al., 2001) Yeast can be used in biological control by presenting antagonistic effect on certain phytopathogenic micro-organisms (Bendo et al., 2009). The control of microbial growth occurs through different mechanisms (Janisiewicz et al., 2000;Droby et al., 2009;Chan et al., 2005). Moreover, other properties such as the lack of production of allergenic spores and antibiotics metabolites, such as the produced by bacterial antagonists, enhance the use of yeasts against some species of pathogenic fungi (Gouvea et al., 2007).
The development of a fermentation process depends on the correct setting of basic factors such as type of microorganism, culture medium, how the process occurs and the steps for product recovery. Suitable operating conditions at bench scale favor obtaining high productivity and yield of the product of economic interest, which helps to expand production to an industrial scale. However, this step is directly related to the interaction of the factors mentioned, as well as to the possibility of reducing the operating costs.
The transfer of a process from bench scale to pilot scale using bioreactors allows obtaining better results, since the optimized conditions favor increased productivity to maximize or minimize, economically and technologically, important variables such as the yield of the process, the concentration of the product and costs (Gomes, 2010).
Thus, the present study objectified to expand the scale of production of biomass of yeasts that have potential for biological control., through changes in the variables: carbon source, agitation and aeration rates.

Microorganisms
The microorganisms used in this study belong to the culture collection of Embrapa Semi-Arid (Petrolina, Pernambuco, Brazil) and were isolated from the natural microflora of mango, melon and grape. One yeast strain was selected from the five tested (L3A, L9, L10, L7K and LF), based in increased biomass production.

Tests on orbital shaker
Nutritional and environmental conditions were evaluated using fermentation processes performed in an orbital shaker (Tecnal, model TE 420), under the following conditions: 30 ± 1 °C at 150 rpm for 32 hours. A synthetic medium was used in this step, with 100 mL in 250 mL erlenmeyer flasks. All flasks were autoclaved at 121°C for 15 minutes.

Influence of synthetic culture medium
The influence of three different synthetic culture media on the biomass production of yeast was evaluated using Sabouraud broth supplemented with 1 % yeast extract and YM (Difco, 2009). Then, 100 mL of each medium were distributed into 250 mL Erlenmeyer flasks and sterilized at 121°C for 15 minutes. The fermentation test was performed at 30 ± 1 °C at 150 rpm for 32 hours.

Essays on bioreactor
The New Brunswinck (Scientific Co.) fermenter, model Bioflo/CelliGen ® 115 with a 3L fermentation vat was used. At this stage, two turbines with six flat blades were used, also known as "Rushton" turbine, to improve the agitation of the reaction medium. Throughout the fermentation process, the pH was monitored using the electrode (Metler Toledo, model 405-

DPAS-SC-K8S).
Culture medium was prepared directly in the vat, and pH was adjusted according to the manufacturer's specifications. At the end of the preparation of the medium, commercial soybean oil was added at a 0.30 % concentration as a foam prevention agent, and then the bioreactor containing the medium was autoclaved at 121 °C for 30 minutes. The simple batch fermentation occurred at 30 ± 1°C for 24 hours at 300 rpm, with aeration rate of 1 vvm.
At first, the behavior of the lineage presenting higher biomass production on orbital shaker was evaluated. In another trial, the influence of the carbon source in biomass production was evaluated by tests that used glucose PA, sucrose PA, crystal sugar and inverted sugar. Once selected the carbon source, the trials were repeated in order to test the concentrations (10 g.L -1 and 20 g.L -1 ). Research, Society and Development, v. 9, n. 4, e174943066, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i4.3066

Determinations
Analytical determinations were performed in triplicate and at established time intervals for sampling: 1) Quantification of cells: biomass produced was determined by spectrophotometer (Varian Model Cary 50 Conc) at 600 nm using a dry weight standard curve previously established; and 2) Quantification of total reducing groups (TRGs): was determined by a colorimetric method using 3,5 dinitrosalicylic acid (DNS) (Miller, 1959); 3) pH control: the pH was monitored in a potentiometric way using a pH meter (Hanna Instruments, model HI 2221).

Results and Discussion
The strain L9 presented the highest biomass production after 96 hours of fermentation, but the strains L7K and L3A also showed a good performance. In addition, yield of total sugars were also the most significant for these three strains. In contrast, the performance of strains L10 and LF was inferior, under the same fermentation conditions, for biomass production and total sugars yield. Biomass production of LF reached only 1.50 ± 0.01 g.L -1 and was associated to the low yield of total sugars (around 7.44 %).
These data are corroborated by the low consumption of available sugars by the strain (32.18 %) after 32 hours. However, it is important to note that L10 strain although producing lower biomass (3.84 g.L -1 ) consumed about 97.88 % of the available sugars at the end of the fermentation process. Therefore, it is necessary a more careful evaluation on the growth rate of the strain and the causes of the low biomass production with the consumption of the available sugars close to 100 %. were related to the greater amounts of biomass produced. Rehm et al. (2005), demonstrated that the pH range that promotes yeasts growth is 2.5-7.0, with an optimum growth at 4.0-5.0.
This fact may explain the amount of biomass produced. Based on previous results, the line L9 showed the greatest production of yeast biomass, being chosen for the subsequent tests. This behavior can be due to the significant percentage of sugar consumed, indicating high conversion of substrate in product. Biochemical and molecular aspects of the adaptation of the yeast during its growth can cause stress to the cells of the micro-organism that induces intracellular changes and their response to the fermentation process (Torrado et al., 2005).
The effect of culture medium on biomass production can be observed in the Table 2 and Figure 1. Data showed that the supplementation of culture medium with a nitrogen source contributed to the increase of biomass in the trials using Sabouraud broth supplemented with 1% yeast extract and YM broth, compared to the growth on Sabouraud broth.
According to Malta (2006), in a study of the propagation of yeast, using yeast extract as a substitute for a conventional nitrogen source, such as ammonium sulfate, it was observed an increase of 2.2 times the cell mass and a yield of 0.22 g/g. Some studies show that nitrogen is an essential element for living organisms and yeast cells can use a wide variety of nitrogenous compounds such as ammonia, amino acids and peptides that induce high growth rates (Terschure et al., 2000;Nitschke et al., 2001).

Figure 1. Relationship between biomass and sugar consumed in different culture media
Consumption of sugars in Sabouraud broth supplemented with 1 % yeast extract was 98.33 % and 85.66 % in YM broth. However, although the sugar consumption was lower in the YM broth, the initial concentration in this medium was 10 g.L -1 compared to the initial concentration of Sabourad broth supplemented with 1 % yeast extract, which was 20 g.L -1 .
Considering the costs of acquiring the culture media, it appears that the YM broth is cheaper ($242,00) compared to the broth Sabourad ($302,00). Although the production of yeast biomass in YM medium has been lower than in the Sabouraud medium supplemented with 1 % yeast extract, it is inferred that this yield may be considered relevant, since a culture Development, v. 9, n. 4, e174943066, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i4.3066 8 medium and cheaply which has in its composition a low concentration of the carbon source, the amount of biomass produced was significant and similar to that of the culture medium which had a high biomass production. According to Reifenberg (1997), the concentration of the carbon source in the medium plays a regulatory role in the metabolism of yeast and contributes as a global regulator of the cell growth mechanism.
The pilot-scale test in a bioreactor was used to evaluate the behavior and performance of the line previously selected in the trials performed in the orbital shaker, selected due to the highest biomass production, the most relevant parameter in this study. In general, assays done in the bioreactor enhanced the performance of the line L9.
An increase in biomass production of 24.12 % was observed compared to the fermentation in the orbital shaker. A reduction of 6.91 % in the percentage of sugar consumed was observed, while the percentage of yield on total sugar and consumed sugar increased 50.15 % and 61.31 %, respectively.
Probably the conditions determined for the bioreactor, such as increased agitation from 150 rpm to 300 rpm and forced aeration, resulted in increased biomass production and, consequently, improved yields. Neves (2003), reported that agitation and aeration are essential for the development of better conditions for the growth of the microorganism in the fermentation processes.
According to Silva et al. (2001), oxygen is important to the whole metabolism, since acts in the generation of energy in the mitochondrial respiratory chain and is essential for obtaining a significant specific growth rate.
As occurred in the trials on orbital shaker, a direct relationship between pH and biomass production was observed, ie, the highest biomass volume (8.03 g.L -1 ) was associated with a highest pH value (6.89).
The effect of carbon sources on the biomass production of the line L9 is shown in Figures 2 and 3. The highest biomass production (6.9 g.L -1 ) at the end of 48 hours of fermentation was recorded when glucose was used as carbon source in the fermentation Development, v. 9, n. 4, e174943066, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i4.3066 9 medium. According to De Leon et al. (2002), yeast are microorganisms able to growth under aerobic and anaerobic conditions and may use a wide variety of compounds as a carbon source, but glucose is the preferred source for the fermentative metabolism.  Development, v. 9, n. 4, e174943066, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i4.3066 In the tests employing crystal sugar and sucrose, biomass produced did not exceed 2.92 and 2.63 g.L -1 , respectively, after 24 of fermentation. Kurzman and Fell (1998), reported that some yeasts do not have the ability to ferment sucrose, using only the glucose present in the culture medium. This could explain the low biomass production observed in this experiment.
The highest consumption of total sugars (91.52 %) occurred in trials using inverted sugar (20 g L -1 ). The use of sucrose resulted in the lowest consumption of total sugars (19.55 %), whereas the presence of glucose caused a consumption of 58.72 %. It was observed that the high consumption of total sugars at the end of fermentation (24 hours) did not cause a high yield of biomass (YR/S: 0.21 g/g) in tests with inverted sugar (20 g.L -1 ). Similar results were observed in tests with sucrose and crystal sugar (YR/S: 0.25 and 0,28 g/g, respectively) at the same time period, for a lower consumption of total sugars. The use of glucose caused total sugars consumption of 58.72 % and 91.78 % for the trials with 10g.L -1 and 20g.L -1 glucose, respectively. The results for biomass production at both concentrations were similar (5.82 and 5.34) suggesting good conversion of carbohydrate into biomass. Taccari et al. (2012), optimized the conditions to produce yeast biomass using a glucose concentration of 20 g.L -1 and observed a biomass production of 5 g.L -1 . In another study, Moares and Bianchi (2013), reported that the biomass produced in 24 hours of fermentation by yeasts isolated from grape skins, using glucose at a concentration of 20 g.L -1 ranged from 3.19 to 3.8 g.L -1 .
The results reported for the tests using inverted sugar at 10 and 20 g.L -1 , showed biomass production of 3.53 g.L -1 and 3.94 g.L -1 , respectively. Despite the low biomass yield compared to that obtained by using glucose, a significant consumption of sugars occurred (87.53 g.L -1 and 91.52 g.L -1 ).
Although glucose PA has shown significant results for the biomass produced, it should be remembered that the high cost of this carbohydrate favors a expensive scale-up process, which suggests the use of inverted sugar as a viable alternative to reduce the operating cost of the process of yeast biomass production. The hydrolysis of the crystal sugar, although causing a higher initial cost, produces an equimolar mixture of glucose and fructose, which are sources more absorbable by the microorganism, which favors the increase of biomass.
The pH is a significant factor for industrial fermentations because of its importance in the control of bacterial contamination and also its effect on yeast growth, fermentation rate and formation of byproducts (2009). Yeasts growth under pH between 3.0 and 8.0. However, pH values ranging from 5.0 to 6.0 are optimal for a high biomass production. The best results were observed using pH in this range, and glucose as carbon source.

Concluison
The use of invert sugar as a carbon source showed a significant biomass production compared to that obtained with glucose, which enables the technique of yeast biomass production with potential to be used in biological control.
In addition, it is observed that it is possible to have a process to produce yeast biomass with a reduced cost and good yield in biomass. However, the relevance of the results is influenced by the choice of carbon source and the increase in agitation and aeration rates.
Future experiments should be carried out in order to observe the potential use of yeasts in the biological control of fungi in food.