Optimization of fermentation conditions for efficient ethanol production by Mucor hiemalis

Microorganism, media, and fermentation

The strain used in this study was M. hiemalis CCUG 16148 (Culture Collection University of Gothenburg, Sweden). This fungus was maintained on agar plates containing glucose (20 g/L), yeast extract (10 g/L), agar (20 g/L), and soy peptone (10 g/L) at 30°C for 5 days and stored at 4°C [14]. The fungal spore suspensions were prepared by addition of 20 mL sterile distilled water to each plate and mixing with an inoculation loop.

Fermentation

All fermentations using M. hiemalis were conducted in 250 mL shaker flasks with 100 mL liquid media. The medium supplemented by 4 g/L yeast extract and 30 g/L glucose. The pH of was then adjusted to 5.5 using 1 M H₂SO₄. The flask was autoclaved for 15 min at 121°C, cooled to room temperature, inoculated with 5 (±1)×10⁶ spores/mL, and incubated at 30°C and 1.6 g under anaerobic conditions [14]. The anaerobic conditions prepared with a loop trap containing glycerol for the gas outlet and two needles for sample removal. The media was purged with pure nitrogen gas at the beginning of the cultivation and during the sampling [14].

The effect of addition of ethanol

The effect of addition of 0.5–12% ethanol on the fungal growth, sugar assimilation, and ethanol production was investigated. An amount of 100 mL solution containing 4 g/L yeast extract and 30 g/L glucose was prepared in 250 mL Erlenmeyer flasks. After autoclave at 121°C for 15 min, the flakes were cooled to room temperature, and predetermined amounts of ethanol were added. The fermentations were then conducted similar to that indicated in Section “Fermentation”.

Enzymatic hydrolysis of wheat flour and effects of glucose concentration

Enzymatic hydrolysis of wheat flour was carried out using two commercial hydrolytic enzymes to convert starch to glucose [15]. α-Amylase (Liquezyme, Novozymes A/S, Denmark) and glucoamylase (Dextrozyme GA, Novozymes A/S, Denmark) were used for the liquefaction and saccharification of wheat flour, respectively. To prepare the hydrolysate, 200 g of wheat flour was mixed with distilled water to achieve the final volume of 1000 mL. The pH of suspension was adjusted to 5.5 by addition of 1 M HCl. It was then placed in a water bath to maintain the temperature at 92°C. Next, 0.2 mL of α-amylase was added to the solution and mixed for 2 h. During this stage, called liquefaction, the enzyme breaks down large chains of starch into smaller ones and maltose. Afterward, the solution was boiled at 95°C for 10 min to deactivate the α-amylase, make it ready for the next stage which was saccharification. The temperature and pH were adjusted to 62°C and 4.5, respectively. Subsequently, 0.2 mL of glucoamylase was added. The second enzyme, glucoamylase, was responsible for saccharification of the short chains of starch and maltose to glucose. After 24 h incubation, approximately 95% of the starch was converted to glucose, resulting in a hydrolysate with 150±3 g/L glucose.

Media containing 10–140 g/L glucose were prepared to investigate the effects of glucose concentration on the growth and ethanol production by M. hiemalis. In these experiments, wheat hydrolysate was used as a source of glucose. The nutrient and fermentation conditions were similar to that mentioned in Section “Fermentation”.

Analytical methods

The liquid samples were analyzed by high-performance liquid chromatography (HPLC, Jasco, Japan). The concentrations of glucose, glycerol, ethanol, and succinic acid were determined using refractive index detector and Aminex HPX-87H with 5 mM H₂SO₄ as mobile phase at 0.6 mL/min and 65°C column temperature for 60 min [16].

For dry weight determination, the biomass was separated by filtering the culture both through the filter (Whatman, 0.45 mm). The biomass was then washed with distilled water, dried at 105°C for 24 h, and cooled in a desiccator before weighing [16].

All the experiments were performed in duplicate and all data reported are the averages of two replications.

Statistical analysis

The central composite rotatable experimental design (CCRD) method was used to determine the effect of three factors, i.e. time, temperature and pH, whereas the response variable was ethanol yield. The object of this work was to find out the best factors for high ethanol production. The central composite design used in this experimental series contained three factors with three levels according to Table 1, in which X₁, X₂, and X₃ were time, temperature, and pH, respectively. In these experiments, the concentration of yeast extract and glucose were 4 and 30 g/L, respectively. It resulted in 20 experiments as shown in Table 1. The number of tests required for CCRD is the sum of 2^k factorial runs with its origin at the center, 2^k axial runs, and numbers of replicate tests at the center, where k is the number of the variables. The values of the variables are coded to lie at ±1 for factorial points, 0 for the center points, and ±2 for axial points [10].

A software package (MINITAB^®) was used to evaluate and fit the second-order model for the independent variables, according to the following equation [13], [17]:

where Y is the dependent or response variable to be modeled, X_i and X_j are the independent variables (factors), and b_i, b_ii, and b_ij are the measures of the X_i, Xi2, and X_iX_j effects, respectively. The variable X_iX_j represents the first-order interactions between X_i and X_j. When the response data are obtained from the experiment, a regression analysis using the least-squares method was carried out to determine the coefficients of the response model, the standard errors, and significances [13]. The effects were considered to be not statistically significant when the p-value was higher than 0.05 at the 95% confidence level [13]. The optimum values of the selected variables were obtained from the estimated variables in the model and by inspecting the response surface contour plots and MINITAB optimizer. The response variable in these experiments was ethanol yield, and the independent variables were time, temperature and pH.

The following equations were used to calculate the biomass and ethanol yield.

where Y, C, V, and M are the ethanol yield (g/g), the ethanol concentration (g/L), liquid volume (L), and the amount of initial glucose (g), respectively.

The inhibitory effect of ethanol on ethanol production

Besides being a product, ethanol is a toxic chemical for all microorganisms. It diffuses through the cytoplasmic membranes of microorganisms and affects their growth and metabolism. The inhibitory effects of ethanol depend on temperature, pH, nutrient availability, and other factors. The strains for commercial ethanol production must be high ethanol tolerant since distillation of dilute ethanol is high energy demanding [17].

To investigate the effect of ethanol on the growth of M. hiemalis, eight experiments were conducted. In all experiments, glucose and yeast extract concentrations were 30 and 4 g/L, respectively, while 0.5, 2, 2.8, 3.3, 5.4, 7.1, 9.5 and 12% ethanol was added.

The effect of adding ethanol on the ethanol production by M. hiemalis is shown in Figure 1A.

Figure 1:

Effect of ethanol concentration in the medium on ethanol production (A), on glucose consumption (B), and glycerol yield (C). In all experiments, the concentrations of glucose and yeast extract were 30 and 4 g/L, respectively.

The consumed glucose and produced glycerol in the solutions are shown in Figure 1B and C).

As shown in Figure 1, the amount of ethanol was increased with time at low concentrations of ethanol. In other words, the fungus could produce ethanol from glucose in the medium containing ethanol. Production of ethanol continued even after addition of 5.5% ethanol to medium; however, when 7.1% ethanol was added, the fungus could not produce ethanol from glucose over the period of time. As shown in Figure 1, the amount of ethanol decreased with time after addition of higher concentration of ethanol (>7.1%). It should be noticed that at 5.5% (v/v) ethanol and higher concentrations, there was no glycerol production by the fungus.

The effect of ethanol on the production of glycerol is shown in Figure 1C. As shown in this Figure, the maximum yield of glycerol was produced in medium containing 0.5% (v/v) of ethanol. When the ethanol percentage was increased, the glycerol production was decreased. Likewise, in experiments with higher than 5.5% ethanol, the fungus did not consume glucose. Additionally, the addition of higher than 7.1% ethanol completely stopped the fungus sugar consumption and ethanol production.

Moreover, in the sample containing 5.5% (v/v) ethanol, no glycerol was produced, ethanol production was insignificant, and glucose was not consumed (Figure 1).

Effect of glucose concentration on ethanol production

Sugar concentration is among the most important factors affecting the yield of ethanol production by microorganisms. The high concentration of ethanol can be obtained by fermentation of high concentration of sugar up to a certain threshold level. However, high concentrations of sugars have negative impact on ethanol production, as result of catabolite or sugar inhibition by reducing the activity of several enzymes involved in the fermentative pathway. The inhibition of ethanol production and growth highly depends on the strain [18].

To study the effect of glucose concentration on ethanol production, different initial concentrations of glucose in the range of 10–140 g/L were used. Hydrolyzed wheat flour was used to supply glucose. Figure 2 shows the produced ethanol and glycerol as well as consumed glucose by M. hiemalis during the fermentation.

Figure 2:

Effect of glucose concentration in the medium on ethanol yield (A), glycerol yield (B), and glucose consumed (C).

In all experiments, yeast extract concentration was 4 g/L and hydrolysate of wheat was used as a source of glucose.

As shown in Figure 2A, at high concentrations of glucose, the inhibitory effect was not observed. For example, in glucose concentration of 140 g/L, the fungus could consume 50 g/L of glucose after 24 h, and production continued further up to 48 h. At glucose concentrations less than 100 g/L, the yield of ethanol was 0.45 g/g after 48 h. According to the Figure 2C, the minimum yield of glycerol was 0.035 g/g of glucose in medium containing 10 g/L of glucose. Also, the maximum yield of glycerol obtained was 0.065.

It can be concluded that at concentrations less than 90 g/L of glucose (i.e. 10, 40, 75 and 90 g/L sugar), the ethanol yields were high and approximately the same. This means that the fungus used in this study has a high sugar tolerant (up to 90 g/L). By increasing the sugar concentration, higher than 90 g/L, the inhibitory effects of sugar was appeared, resulting in the lower yield of ethanol.

In a previous study, Radmanesh et al. [15] evaluated the inhibitory effects of ethanol on ethanol production by M. hiemalis; however, in their experiments, they started fermentation by 1 g/L of the fungus biomass, while all fermentation in this study was started with the fungal spores. The comparison of their results with the current work results indicated that the fungus can tolerate higher sugar concentration when fermentation is started with biomass, rather than the spores. In other words, M. hiemalis spores are more sensitive to high sugar concentration than its biomass. This was also observed other strains of Mucor, e.g. Mucor indicus [19].

Effects of fermentation conditions on ethanol production

The effects of pH, time, and temperature on ethanol yield were investigated, and the results are summarized in Table 2. Furthermore, surface and contour plots for the determination of effect of pH, temperature, and time on ethanol yield by response surface method are shown in Figure 3. As shown in this figure, ethanol yield was increased by increasing temperature up to 30°C, while it was decreased by further increasing temperature to 35°C. The yield of ethanol was increased with time and pH. The maximum yield of ethanol occurred after 36 h. The maximum ethanol yield was obtained at the highest time and medium temperature and pH.

Figure 3:

Surface and contour plots for determination of effect of parameters on ethanol yield by response surface method.

The parameters were pH, temperature, and time. The temperature and time are in terms of (°C) and hour. The lines in the contour plots are related to the ethanol yield. (A) pH vs. T, (B) time vs. T, (C) pH vs. time.

The yield of ethanol was between 0.08 and 0.48 g/g, whereas glycerol yielded between 0.004 and 0.06 g/g. In test 7^th and 9^th, the maximum ethanol yield of 0.48 g/g was obtained, where the maximum glycerol yield of 0.055±0.005 g/g was obtained. Furthermore, in these tests, glucose was completely consumed, and the yield of biomass was 0.01 g/g, which was the minimum among all experiments. The minimum ethanol yield was obtained at low temperature and pH. Ethanol yield of 0.08 g/g was obtained at 25°C, the least among all experiments.

Additionally, the yield of biomass was between 0.01 and 0.16 g/g. According to the Table 2, the highest yields of ethanol, biomass, and glycerol were also obtained as 0.48, 0.16, and 0.06 g/g, respectively.

Moreover, the results of statistical analysis, including the estimated values of factors, coefficient, interactive terms, t from student’s t-test, and p-values are shown in Table 3. The results indicated that all of the terms had significant interactions (p<0.05).

Substitution of coefficient calculated and response variables in equation (1) resulted in the following empirical equations for ethanol yield:

The coefficients of equation (3) can be obtained from Table 3. The predicted values obtained from the model equation (3) showed very good agreements. The suitability of the fitness was checked by determination coefficients (R²), which was 99.3% for the yield of ethanol. The R² statistic indicates the percentage of the variability of the optimization parameter that is explained by the model, and therefore, 0.7% of the total variation was not explained by the models developed for the yield of ethanol.

Millati et al. [5] cultivated M. hiemalis on glucose at 37°C and pH 5±0.7 for 7 days. After about 48 h fermentation, they obtained the maximum ethanol and glycerol yields of 0.39 and 0.042 g/g, respectively. They also reported the biomass yield of 0.09 g/g after 7 days. The results are generally in agreement with the current study results and the minor lower yield of ethanol and glycerol might be due to the cultivation at higher temperature applied.

Radmanesh et al. [15] cultivated M. hiemalis at pH 5.5 at 37°C and modeled ethanol production. They obtained the highest ethanol yield of 0.44 g/g and did not report the glycerol yield. The results of current work indicated that it is possible to improve the ethanol yield up to 0.48 g/g by optimization of the fermentation conditions. Sues et al. [20] optimized ethanol production by M. indicus. They found the highest ethanol yield of 0.46 g/g at 30°C and pH 5.6 after 24 h cultivation under anaerobic conditions. Thus, compared with M. indicus, M. hiemalis produced the higher yield of ethanol.