Study of the growth of two yeast strains (Yarrowia lipolytica and Pichia guilliermondii) in an ammonium formate solution of rubber effluents

doi:10.21203/rs.3.rs-2503415/v1

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Study of the growth of two yeast strains (Yarrowia lipolytica and Pichia guilliermondii) in an ammonium formate solution of rubber effluents

https://doi.org/10.21203/rs.3.rs-2503415/v1

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The growth of microorganisms is influenced by several physico-chemical parameters that need to be controlled before starting a biological treatment plant for better process efficiency. In this study, the influence of temperature (25-40 °C), pH (4-8), and ammonium formate concentration (1.59-7.94 mM) on the growth of two yeast strains (Y.lipolytica and P.guilliermondii ) examined in a batch process. Temperature has a direct impact on the kinetic growth parameters with an activation energy (Ea) of 14.3 kcal/mol and R² 0.95 for Y.lipolytica and 12.5 kcal/mol and R² 0.97 for P.guilliermondii, what indicating a dominant biological regime. The yeast strains are acidophilic with a maximum growth pH between pH 5 and 6.

Yarrowia lipolytica

Pichia guilliermondii

ammonium formate

rubber wastewater

Batch bioreactor

Ammonium formate is produced in the rubber industry by the reaction of formic acid, used in the factory to coagulate the latex, and ammonia, which is incorporated into the latex in the field to prevent premature coagulation (Iagba et al., 2008). Ammonium formate is an ammonium salt with exposure effects that vary with concentration. Transient and reversible health effects are observed at 3.7 g/l. From 41 g/L, the effects become adverse and irreversible (severe irritation and permanent damage to the eyes, irritation of the mucous membranes and respiratory tract, stomach pain and difficulty breathing and finally gastrointestinal irritation) and death occurs above 240 g/L, the lethal dose (SCAPA, 2016; USEPA, 2016;). Furthermore, when this compound dissolves in water, it produces two species (ammonium ions and formate ions) that are environmentally toxic either directly or through their metabolites. Water containing the ammonium ion causes the formation of carcinogenic nitrosamines, the formation of methemoglobin in infants, and the inhibition of certain digestive and respiratory tract pathways (Nsoe et al., 2016; Sulaiman et al., 2010; Arimoro, 2009; Bourgard, 2004). Environmental eutrophication of mangroves causes dissolved oxygen depletion and consequent death of aquatic biological life (Dibyendu and Homagn., 2016; Tekasakul, 2010). Increased ammonium ion concentrations in the environment cause long-term imbalance in the nitrogen cycle and accelerated denitrification, which can increase concentrations of nitric oxide and nitrous oxide, both of which contribute to ozone depletion (Viard et al., 2013). The reduction of formations produces CO₂, which is responsible for more than 60% of the greenhouse effect. Given all of the health and environmental concerns associated with this compound, it is critical to treat the effluent from rubber latex coagulation before it is released into the environment. Lagoons and oxidation pits are biological processes to treat rubber industry effluents without considering abiotic and biotic factors (Ndi et al., 2016, Mitra et al., 2010; Tekasakul., 2010). The contact time between the microorganism and the pollutant load therefore requires a long residence time, release of odors and non-compliance with legal discharge limits. Therefore, it is important to consider these factors. Abiotic and biotic factors influence biological wastewater treatment by promoting or inhibiting the development of microorganisms responsible for the biodegradation of pollutants (Dehghani and Hassan., 2013). Controlling and understanding these factors is beneficial both in the design of wastewater treatment plants and in improving and understanding microbial activity to ensure purification performance (Milton, 2009; Songming and Shulin 2002; Bo and Swantje, 1998; Harvey and George 1991). The main factors affecting treatment efficiency are pollutant concentration, temperature and pH. (Fqih et al., 2000; Nedwell, 1999). The main factors affecting treatment efficiency are pollutant concentration, temperature and pH. (Fqih et al., 2000; Nedwell, 1999). Every microorganism has a pollutant tolerance threshold. Above this point, the pollutant becomes an inhibitor, slowing the growth of microorganisms. For this reason, understanding the concentration range is crucial in order to optimize treatment (Dutta et al., 2014). The same applies to pH, which affects both the thermal resistance of the yeast membrane and the membrane structure. In fact, a change in pH affects membrane potential by decreasing or increasing nutrient diffusion at the cell membrane level. The ionic state of nutrients is also affected by pH, resulting in the Donnan effect, which reduces nutrient permeation across the membrane (Dehghani and Hassan., 2013; Xiang et al., 2012). Temperature can act as a catalyst or inhibitor depending on the environment. Yeasts develop resistance and become spores at low temperatures, which reduces their metabolism and thus the cleaning performance of the stations. On the other hand, at high temperature, there is reversible dissociation and irreversible denaturation of proteins at the membrane level, which can lead to yeast death and low purification yield (Milton, 2009; Fqih et al., 2000). From now on we will study the effect of these three parameters (ammonium formate concentration, pH and temperature) to determine the optimal conditions for the growth of yeast species (Yarrobia lipolitica and Pichia guillermondii separately from Nsoe et al., al., (2013) to determine. from nature, rubber plant waste and has demonstrated the ability to grow in rubber ducts.

2.1. Yeast inoculum.

The yeast strains used in this work were isolated from rubber industry waste thanks to the Nsoe protocol (Nsoe et al.2013). Microbial properties are listed in Table 1.

Table 1

Characteristics of yeast strains (Nsoe et al., 2018)
Code	Yeast colour	Zeta Potential pH (5,7–7)	Yeast shape	Yeast size (µm)	Growth in selective media
Code	Yeast colour	Zeta Potential pH (5,7–7)	Yeast shape	Yeast size (µm)	Acid medium	Alkaline medium
Yarrowia lipolytica	Red	-25,3 to -37,8	Ovoid	0,3-150	+	+
Pichia guilliermondi	White	-27,8 to-30,5	Ovoid	0,2-100	+	+

Yeast was cultivated on the following growth medium (Atagana, 1996): meat extract 1 g. L^− 1 (Sherlan rf 07–075, Spain), yeast extract 2 g. L^− 1 (OXOID code L21, England), peptone 5 g. L^− 1 (Liofilchem rf 610038, Italy), sodium chloride 5 g.L^− 1 (Jeulin rf 107115, France) at pH = 6.5.

2.2. Synthetic influent.

A synthetic influent was created to ensure the concentration common in the rubber industry. Ammonium formate is the sole source of carbon and nitrogen. This was added to cover the varying nitrogen content: from 1.59 to 7.94 mM (22.3 to 111 mgN.L-1). Ammonium formate was mixed with mineral salt medium (in g.L^− 1): MgSO₄ (0.2), CaCl (0.02), KH₂PO₄ (60), K₂HPO₄ (14). A stock solution of microelements (in g.L^− 1): ZnSO₄(10.90), FeSO₄(5), MnSO₄(1.54), CuSO₄(0.39) was prepared and added to the influent at 0.1% (v/v ). Then 0.25 g of L^− 1 chloramphenicol was added to inhibit bacterial growth and the synthetic influent was sterilized at 110°C for 10 minutes.

2.3. Experimental set–up: Batch bioreactor.

Figure 1 shows us the batch bioreactor assembled for this experiment, it consists of several 1L bioreactors (with a usable volume of 0.8L) that were used and immersed in a water bath to keep the temperature at 25C. Aeration was supplied thanks to an air compressor (GIANESSIEDILIO, NML 58629, LT 100, ATE 12TEMPA, Italy) to ensure that dissolved O2 was not a limiting factor for respiration and biomass growth. To reduce bacterial contamination and CO2 levels, the air inlet is subjected to a series of washes containing 1mM sodium hydroxide and 1mM hydrochloric acid. Then, the air outlet was connected to a 500 mL vial containing a 0.5 mM sodium hydroxide solution to quantify the CO2 released during the degradation of ammonium formate

2.5.2. Influence of ammonium formate concentration on growth

Figure 1 shows us the batch bioreactor assembled for this experiment, it consists of several bioreactors of 1 L (with a useful volume of 0.8 L) used and immersed in a water bath to keep the temperature at 25°C. Aeration was supplied thanks to an air compressor (GIANESSIEDILIO, NML 58629, LT 100, ATE 12TEMPA, Italy) to ensure that dissolved O₂ was not a limiting factor for respiration and biomass growth. To reduce bacterial contamination and CO₂ levels, the air inlet is subjected to a series of washes containing 1mM sodium hydroxide and 1mM hydrochloric acid. Then, the air outlet was connected to a 500 mL vial containing a 0.5 mM sodium hydroxide solution to quantify the CO₂ released during the degradation of ammonium formate.

2.5.3.1. Activation energy

The effect of temperature on microbial growth was evaluated via the activation energy obtained using the Arrhenius equation (Kirchman, 2012; Attilio and Jose, 2001). This relationship describes the general dependence of the rate of a reaction on the temperature and is given by Eq. 1.

\({{\mu }}_{{m}{a}{x}}={{\mu }}_{0}{{e}}^{\left(\frac{{-{E}}_{{a}}}{{R}{T}}\right)}\) Eq. 1

With:

E_a: Activation energy of microbial growth, (kcal/mol); µ₀: pre-exponential factor (h^− 1) ; R : gas constant (kcal/mol. K); T: Temperature (K).

2.5.3.2. Q10 Coefficient

This parameter represents the increase in reaction rate when there is 10°C increase in temperature. The value of Q₁₀ is calculated using Eq. 2 (Urbano et al., 2005):

\({{Q}}_{10}={\left(\frac{{{K}}_{2}}{{{K}}_{1}}\right)}^{\left(\frac{10}{{T}2-{T}1}\right)}\) Eq. 2

T₂ : Upper temperature

(°C); T₁ : Lower temperature

(°C); µ₂ : Higher reaction speed

; µ₁ : Lower reaction speed

2.5.4. Influence of hydrogen potential (pH) on yeast growth.

The study of the influence of pH was carried out in a pH range of 5 to 8. Adjustment of pH of the medium was done with hydrochloric acid solution (1M HCl) or a 1M NaOH solution deep.

3.2.1. Yeast kinetic growth

Figures 2 and 3 individually show the evolution of the population of strains of Yarrowia lipolytica and Pichia guillermondii as a work of time. These engine bends all have classic yeast development profiles for both strains (initial stage, accelerated stage, decaying stage, stationary stage).

Yeast growth varies with the concentration of ammonium formate independent of the strain. In the two strains, two phenomena were observed when the formate concentration was greater than 3.17 mM; as the latency period increases, the growth of the yeast slows down. According to Amrouche et al. (2011), the increase in the latency period is an adaptation of the yeast to high concentrations of the substrate. This increase in latency can also be caused by the obstruction of the enzyme's catalytic center by the excess substrate, or alternatively, the substrate can lodge in the active site with an abnormal orientation, preventing the reaction from proceeding; This is the reason for the low yield of yeast production caused by low number of metabolized substrates due to inhibition. On the other hand, when the formate concentration is less than or equal to 3.17 mM, vigorous growth of microorganisms was observed due to availability of the substrate. However, at concentrations less than 3.17 mM, this growth is weaker than that obtained from the concentration of ammonium formate at 3.17 mM. This is because the 3.17mM maximum concentration is the concentration for optimal yeast growth.

Thus, the ammonium formate concentration at 1.59 mM is insufficient to ensure maximum yeast growth due to substrate depletion. In order to better visualize the influence of the concentration of ammonium formate on the growth of yeast, a representation of the influence of the concentration on the maximum growth rate is created.

I.3.2.2. Maximum growth rate of yeasts at different concentrations of ammonium formate.

The Pichia guilliermondii strain shows a maximum development rate (UFC/mL/h-1) of 18*104. This speed is 1.21 more remarkable than the maximum of strain Yarrowia lipolytica which is 15*104. This may be due to a strong adaptation of the Pichia guilliermondii strain to ammonium formate, which shortens the adaptation time, or it may be due to a less complex digestive system compared to the Yarrowia lipolytica strain. To confirm this, we determine the duplication factor.

I.3.2.3. Yeast growth multiplication factor as a function of ammonium formate concentration.

Although the maximum growth rate of Pichia guilliermondii is higher than that of Yarrowia lipolytica, Yarrowia lipolytica has been found to have a 60% higher biomass production rate than Pichia guillermondii. This shows us that Y. lipolityca reproduces rapidly compared to P. guillermondii. The optimum growth rate and multiplication factor for both strains is 3.17 mM, which is higher than the maximum concentration found in rubber mill effluents. In order to investigate the influence of other parameters, this concentration is recorded and kept constant.

I.3.3. Impact of temperature on yeast growth.

Temperature is one of the important factors to consider when studying yeast growth. It can actually modify growth kinetic profiles; therefore we have highlighted its influence on specific growth rate, activation energy and Q₁₀.

I.3.3.1. Yeast growth kinetics as a function of temperature

For the two yeast strains studied, we followed the growth of the yeasts throughout the biodegradation of ammonium formate. As shown in Figs. 8 and 9 for the two yeast strains.

Figures 6 and 7 show the obtained biomass production profiles as a function of time for temperatures of 25, 30, 35 and 40°C. Although the growth of the biomass presents the same phase (starting phase, acceleration phase, deceleration phase, stationary phase), regardless of the temperature and the stress between 25 and 30 ° C, the temperature has a positive influence on the yeast growth due to a latency time that is independent of the yeast strain is almost zero. In this temperature range, the production of biomass is maximum, but varies from strain to strain. This observation confirms that the yeasts were activated upon inoculation and that the preculture conditions were satisfactory. The heat from the environment provides additional energy that facilitates enzymatic reactions, leading to an increase in biomass and consequently a reduction in the residence time of the pollutant in the bioreactor. On the other hand, between 35ºC and 40ºC we observe a slowdown in the production of biomass, with a latency that increases with temperature and differs from strain to strain. This rather long latency period may reflect an increase in biodegradation time and poor biodegradation of the organic matter. These results can be compared to those obtained by various authors on the effect of temperature on yeast growth (Torija et al., 2002). Lucero et al., (2000) believe that increasing the temperature leads to damage to the structure of the cell membrane and consequently to a reduction in its transmission properties. Denaturation of the secondary and tertiary structures of the enzyme can also occur.

I.3.3.2. Influence of temperature on specific growth rate

It can be seen that the specific growth rates are strongly influenced by temperature. Regardless of the strain, the specific growth rate decreases with increasing temperature. With maximum values at 28°C in the Pichia guilliermondii strain (0.112 h-1) and at 25°C in the Yarrowia lipolytica strain (0.102 h-1). These results agree with the work of Torija et al. (2002), who confirm the negative effects of temperature on yeasts. For a better cleaning performance and for the cultivation of the yeast, it is advisable to work in the range of 25 to 30°C.

I.3.3.3. Activation energy

The Arrhenius equation describes the general dependence of the rate of a reaction on temperature (Cisse et al., 2009) as shown in Fig. 9.

Table 2

Activation energy (Ea), and regression coefficient (r²) for the two yeast strains.
Microorganismes	Ea (kcal/mol)	r²
Yarrowia lipolytica.	11,3	0,95
Pichia guilliermondii.	8,3	0,97

We determined the parameters of the Arrhenius equation for the two strains using the equations on the right side of Fig. 9, which represents the natural logarithm of the maximum growth rate (biomass production) at different temperatures. The values of the activation energy (Ea) and the correlation coefficient r2 are recorded in Table 2. The activation energies obtained are 11.3 and 8.3 kcal/mol, respectively, for the strains Yarrowia Lipicica and Pichia guilliermondii with r² of 95 and 97. From the values it is concluded that Pichia guilliermondii is less sensitive to temperature than Yarrowia lipolytica. The latter requires more energy to carry out its metabolic reactions. With an activation energy value equal to or greater than 12 kcal/mol, the activation process is in a biological regime (Sanchez et al., 2004; Serra et al., 2005). We conclude that we are in a biological regime for Yarrowia lipolytica and in a diffusive regime for the strain Pichia guilliermondii.

I3.3.6. Q10 factor

In the same way as for the activation energy, the value of Q10 can be used to know if the process is physical (Q10 ≤ 1) or biochemical (Q10 ≥ 2) (by diffusion or biological). The Q10 coefficient is also a useful tool to indicate the sensitivity of the response to an increase in temperature within a defined range by measuring changes in growth rate, as shown in Table 4.

Table 3

Valeur du Q₁₀ pour *Yarrowia lipolytica* et *Pichia guilliermondii*
yeast	Q₁₀ (entre 25–35°C)	Q₁₀ (entre 30–40°C)
Yarrowia lipolytica	5,07	3,32
Pichia guillermondii	1,88	1,19

Table 3 above shows that the Q10 factor is a function of strain and temperature. Between 25 and 35°C the Q10 value is 1.88 for Pichia guilliermondii and 5.07 for Yarrowia lipolytica. While between 30 and 40 C this value decreases for the two strains Pichia guilliermondii (Q10 = 1.19) and 3.32 for Yarrowia lipolytica. According to Apple et al., (2006), Sand-Jensen et al. (2007), Q10 values are higher at low temperatures because under such conditions the biochemical reactions involved are limited by a decrease in enzymatic activity. On the other hand, at high temperatures (above the threshold temperature) the value of Q10 decreases and under these conditions a physical limitation occurs, for example the decrease in oxygen diffusion.

I.3.4. Influence of pH on yeast growth.

Figures 10 and 11 show the influence of pH on yeast growth as a function of time. It can be seen that the two yeast strains show maximum growth between pH 5 and 6. This means that at this pH the solubility of nutrients is maximum, the transport of nutrients inside the cell, the enzymatic reactions as well as the surface phenomena are favored by modifying the configuration of the active sites of the cell wall (Papagianni, 2004). On the other hand, at pH 8, a growth slowdown is observed, which can be caused by changes in the conformation or polarity of the membrane (transport systems) or the substrate molecules (proteins), or else by the denaturation of enzymes.

The ability of Yarrowia lipolitica and Pichia guillermondii to grow in a medium rich in ammonium formate was studied. It shows that the concentration of ammonium formate is observed at a maximum growth of P.guilliermondii and Y.lipolytica of 3.17 mM, which is higher than the concentrations of ammonium formate found in the rubber effluent. Temperature has a direct impact on yeast growth with a maximum between 25 and 30°C. Yeast strains are acidophilic with a maximum growth pH between pH 5 and 6. The formate molecule can be either a substrate or an inhibitor depending on the concentration. In order to know the molecule responsible for the inhibition and the types of inhibition, it would be advisable to study the biodegradation kinetics of the formate and the formate present in the medium.

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No competing interests reported.

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Study of the growth of two yeast strains (Yarrowia lipolytica and Pichia guilliermondii) in an ammonium formate solution of rubber effluents

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Abstract

Figures

Introduction

Materials And Methods

2.5.2. Influence of ammonium formate concentration on growth

2.5.3.1. Activation energy

2.5.3.2. Q10 Coefficient

Results And Discussion

3.2.1. Yeast kinetic growth

I.3.3.1. Yeast growth kinetics as a function of temperature

I.3.3.2. Influence of temperature on specific growth rate

I.3.3.3. Activation energy

Conclusion

References

Additional Declarations

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