The Effects of Dilution,Aeration,and Agitation on Fungal Cellulase and Xylanase Production in the Fermentation Media Based on Distillers Dried Grains with Solubles Using Stirred Tank Bioreactors

Abstract

Fungal cellulases and xylanases are commonly used for the biological hydrolysis of lignocellulosic biomass into simpler sugars, which can then be converted into various value-added products like bioethanol. However, these enzymes may be costly upon production strategy. Therefore, the enzyme production process should be more efficient by utilizing economical and highly fibrous feedstocks and other nutrients. Distillers dried grains with solubles (DDGS) is a coproduct of corn ethanol generation and can be considered an appropriate feedstock. Regarding the widely used submerged fungal enzyme production process, low activity is the most prominent challenge. Therefore, this study is undertaken to improve further the submerged fungal fermentation process to lead to higher fungal cellulases and xylanases. Dilute acid-hydrolyzed DDGS medium with solid DDGS particles (i.e., mostly cellulosic fraction) was used at varying dilutions to evaluate the effect of fermentation system parameters on enzyme productions in 2-L bench-top bioreactors by Aspergillus niger (NRRL 330). Namely, the effects of aeration and agitation were explored for each dilution factor for both cellulase and xylanase productions. A sharp increase in cellulase and xylanase was observed with higher agitation and aeration rates. The highest cellulase activity (0.76 IU/mL) was obtained on Day 7 when low dilution (2X) and higher agitation (500 rpm) and aeration rates (1 vvm) were used. On Day 7, the xylanase activity was 28.10 IU/mL implying simultaneous production of both enzymes. A higher xylanase activity of 29.04 IU/mL was achieved at the same reactor conditions on day 8 of fermentation. In conclusion, this study confirms that dilution, aeration, and agitation rates enhance enzyme production in the early phases of fungal fermentation. This study is certainly a step forward for the production of fungal cellulase and xylanase enzymes using DDGS as feedstock.

Introduction

Due to the high consumption of fossil fuels in the current industrial era, the demand for renewable energy resources is continuously growing. The transportation sector occupies approximately 15% of the total greenhouse gas emissions.¹ This sector also demands liquid renewable energy sources, which are mostly met by bioethanol at the moment with a 10–15% blending ratio. Bioethanol production has been estimated to be at least 140 billion liters.¹ In the US, first-generation (1G) bioethanol is produced from corn and mostly (89.4%) by a process called dry-grind ethanol production.² Currently, the United States is the top producer of bioethanol worldwide with 56% of total production capacity.² A coproduct of 1G ethanol production process is distillers' dried grains with solubles (DDGS), which is one-third of the grains left after most of the starch is converted to ethanol by yeast.³ The US produces more than 44 million metric tons of DDGS each year.⁴ DDGS is high in dietary fiber such as acid detergent and neutral detergent fibers along with significant protein and fat contents.

Since ethanol is one of the most attractive alternative fuels in the transportation sector, various strategies have been proposed to improve the bioethanol production process. One prominent strategy proposed so far is the use of a lignocellulosic or non-food fraction of crops as raw material for the production of ethanol, which is known as second-generation (2G) bioethanol.⁵ However, a major bottleneck in this process is the need for economical hydrolytic enzymes mainly cellulases and xylanases, which are needed to convert the complex lignocellulosic biomass into simpler sugars. These sugars can then be converted into bioethanol. To produce those enzymes, a feedstock, which shall be economical and high in fiber and protein contents, is needed. Therefore, DDGS is an ideal feedstock, in this regard, as it will facilitate the onsite enzyme production and decrease the economic burden of transporting the feedstocks over longer distances.

While the use of cellulases and xylanases provides a solution to the industrial adaptation of 2G ethanol production, currently these enzymes are not widely used, due to low activity levels and high cost.⁶ By an estimate, only 6% of the ethanol comes from lignocellulosic feedstock, which gives an idea about the use of such enzymes in the ethanol industry.² There are mainly two types of fermentation modes adaptable to enzyme production. Solid-state fermentation (SSF) is proven to be better in high enzyme activities, but it hinders production at industrial scales. Most microbial strains, especially fungal strains such as Aspergillus niger and Trichoderma reesei, which are shown to produce high activities of such hydrolytic enzymes, prefer solid substrate supports. On the other hand, these microbial strains show relatively lower enzyme activities in submerged fermentation.⁷

If these fungal strains are cultivated in submerged fermentation in bioreactors with aeration and agitation systems, they stick to solid surfaces such as the impeller, aerator, and agitation shaft in the bioreactor, making semi-solid pellets that differ from batch to batch. This observation was also noted by Ramamoorthy et al., who observed that the fungal strain, Trichoderma harzanium (ATCC 20846), was “harboring to the baffles, agitator, and the impeller shaft, and continuing a partial solid-state growth”.⁸ The fungal cells in the liquid broth surrounding this partial solid-state growth also show dense cell growth, forming a heterogeneous matrix in the reactor. Cellulase activities are also lowered by agitation rates and shear stress to the mycelial cells created by high agitation rates.⁹ The high agitation rates, however, are needed to distribute the nutrients and oxygen in the media.

One way to solve the problem of dense fungal pellets hindering the release of extracellular hydrolytic enzymes is the use of a semi-solid type of fermentation mode where small solid particles are present in liquid media. This can serve two purposes: first is that smaller solid particles can disrupt the fungal pellet formation and second, the fungal cells can stick to such mobile solid particles as the solid support without sticking to the surfaces inside the bioreactor. For this purpose, submerged fermentation media including small particles (i.e., cellulosic particles) would be ideal. Therefore, DDGS, after pretreatment, can be used as such media without filtering the rest of the DDGS particles out. The DDGS particles in the media would not only provide a hindrance to the fungal pellet formation but also help to secrete high enzyme activities as these particles are made of cellulose and partly hemicellulose fibers.

Hence, this research is undertaken to utilize a semi-solid media or a liquid media with solid particles of DDGS at different levels of dilutions to produce cellulase and xylanase enzymes. The overall objective of this research was to study the effect of aeration, agitation, and dilution rates on nutrient consumption and enzyme production in bench-top bioreactors.

Materials and Methods

EXPERIMENTAL DESIGN

The objective of this study was to assess the effect of operational parameters on cellulase and xylanase production. The independent variables studied were the aeration rate (0 and 1 vvm), agitation rate (100 and 500 rpm), and dilution rate (2X and 4X dilution). A fractional factorial design with treatments was performed as shown in Table 1.

Table 1.

Levels of Dilution Factor, Aeration, and Agitation Used for Fractional Factorial Design to Evaluate Cellulase and Xylanase Production

FACTOR	LEVELS
FACTOR	1	2
A: DF	2	4
B: Aeration (vvm)	0	1
C: Agitation (rpm)	100	500

All the experimental conditions were repeated 3 times.

MICROORGANISM AND INOCULUM PREPARATION

The microbial strain used for this study was Aspergillus niger (NRRL 330), mainly because of its higher enzyme activity levels as reported in a previous study.¹⁰ A. niger (NRRL 330) was obtained from Agricultural Research Service (ARS) Culture Collection (Peoria, IL). For the revival, the obtained spores were suspended in sterile 0.1% (w/v) Tween 80 solution and were then spread on potato dextrose agar (PDA; VWR International, Radnor, PA), and incubated at 30°C. Spores were harvested using sterile 0.1% Tween 80 solution and stored in 20% (w/v) glycerol (VWR) at -80°C until use.

To be used as the inoculum, A. niger (NRRL 330) spore suspensions were spread over PDA plates before each fermentation run. After 5 to 7 days of incubation at 30°C, plates were flushed with 10 mL of 0.1% Tween 80 solution, and the spores were homogenized by a sterilized glass hockey stick. The suspension was then diluted to give an OD₆₀₀ of approximately 0.9. This diluted spore solution was then used as the inoculum.

DISTILLERS DRIED GRAINS WITH SOLUBLES (DDGS)

Distillers dried grains with solubles (DDGS) was provided by Pennsylvania Grain Processing, LLC^® Ethanol Plant (Clearfield, PA). The protein, fat, and fiber contents of DDGS were provided by the manufacturer as shown in Table 2.

Table 2.

The Chemical Composition of Distillers' Dried Grains with Solubles (DDGS) Provided by Pennsylvania Grain Processing

COMPONENT	AS RECEIVED (minimum%-maximum%)	DRY WEIGHT (minimum%-maximum%)	METHOD OF DETECTION
Dry Matter	86.0-88.1	–	NFTA 2.2.2.5
Protein (crude)	26.3-28.1	30.6-32.2	AOAC 990.03
Fat (crude)	6.14-8.01	7.03-9.09	AOAC 945.16
NDF	28.4-32.7	32.2-37.4	AOAC 2001.11
ADF	11.2-11.6	12.7-13.3	Ankom Tech. Method
Fiber (Crude)	7.82-8.12	8.88-9.30	AOCS BA 6A-05
Hemicellulose^*	17.2-21.1	19.5-24.1	Calculation
Ash	4.36-4.66	4.95-5.42	AOAC 942.05

ADF: Acid detergent fiber; NDF: Neutral detergent fiber: AOAC: Association of analytical chemistry; NFTA: National Forage Testing Association; ^*Hemicellulose component = NDF-ADF

PRETREATMENT AND PREPARATION OF DDGS-BASED MEDIA

Dilute acid hydrolysis was used for pretreatment and partial hydrolysis of DDGS to simpler compounds, as suggested by our previous research.¹¹ The conditions for the pretreatment included 5% (w/v) sulfuric acid, 20% (w/v) solid load, and 30 min of treatment time at 121°C in an autoclave (Model Beta Star, RV Industries, Honey Brook, PA). The resulting slurry was not filtered, but the pH of the entire mix was adjusted to 5.00 ± 0.20 by adding 10 M NaOH (VWR).

BIOREACTOR SET-UP

The prepared acid-hydrolyzed DDGS slurry was then added to the 2-L glass vessel with a 1.5 L working volume (Biostat B-Plus bioreactor, Sartorius, Allentown, PA). The reactor vessel was then sterilized in an autoclave (RV Industries) for 45 min and cooled to 30°C. The prepared A. niger (NRRL 300) spore suspension (10%) was added aseptically to the bioreactor as the inoculum. The reactor's temperature and pH were maintained at 30°C and pH 5, respectively, for 9 days of fermentation. Samples were taken daily and stored at -20°C until analysis. The aeration and agitation along with temperature and pH were controlled automatically by Sartorius B-Plus bioreactor instrumentation according to the experimental design.

SAMPLE ANALYSIS

All samples were centrifuged at 1,510 x g to remove the solid DDGS particles from the liquid broth. The supernatant was then collected and further filtered using 0.45 μm PTFE filters (VWR). The cell-free broth was further diluted for the sugar and enzyme analyses as described below.

Simple sugar analysis

The monosaccharides and other reducing sugars (simple sugars) were tested using the dinitrosalicylic (DNS, VWR) method.¹² Three milliliters of DNS solution were mixed with an appropriately diluted sample and boiled for 15 min, along with blanks and glucose (0.1–0.6g/L) standards. The glucose standard curve was then used to calculate the total sugars in the media.

Cellulase analysis

Cellulase activity in terms of international units (IU/mL) was assayed by diluting the samples according to the concentration of reducing sugars in each sample in citrate buffer at pH 4.8 and adding 1x6 cm (∼50 mg) long strips of Whatman filter paper (GE Healthcare, Chicago, IL) to each reaction tube.¹² Substrate blanks were prepared by diluting the samples with the citrate buffer without the addition of filter paper and the enzyme blank contained filter paper and citrate buffer. All sample mixtures as well as the substrate blanks, enzyme blanks, and glucose standards were incubated at 50°C for one hour. Immediately after incubation, 3 mL of DNS solution was added to each tube. All tubes were then boiled for 15 min.

Sugar levels were then determined by measuring the absorbance values at 575 nm using a spectrophotometer (Evolution 21, Thermo Scientific, Oakwood, OH). The activity levels were estimated by subtracting blank tubes from the reaction mixture tubes using the established standard curve for conversion into corresponding glucose concentrations in g/L. This concentration was then converted into the unit of enzyme activity by using the following equation: $\frac{I U}{m l} = \frac{G l u c o s e C o n c .}{μ m o l e (g l u c o s e) * t i m e}$ (Equation 1)

One unit (IU) of cellulase activity is defined as the amount of enzyme that releases one micromole of glucose per minute under assay conditions.

Xylanase analysis

Xylanase analysis was performed in the acetate buffer at pH 5. After appropriately diluting samples based on the sugar concentration in citrate buffer, 1 mL 0.5% (w/w) xylan solution (Crescent Chemicals, Islandia, NY) was added to the sample tubes. The substrate blank did not have xylan solution and the enzyme blank did not contain any sample. The reaction tubes along with blanks and standards (0.1–0.6 mg/mL xylose) were incubated at 50°C for 30 min. Immediately after the incubation, 3 mL of DNS solution were added to each tube and the tubes were boiled for 15 min. The absorbance levels of standards with the tubes were measured at 575 nm.

This concentration was then converted into the unit of enzyme activity by using the following equation: $\frac{I U}{m l} = \frac{X y l o s e C o n c .}{μ m o l e (x y l o s e) * t i m e}$ (Equation 2)

One unit (IU) of enzyme activity for xylanase is defined as the amount of enzyme that liberates one micromole of xylose per minute under assay conditions.¹²

STATISTICAL ANALYSIS

Two-Way Analysis of Variance (ANOVA) was performed to evaluate the effect of each experimental combination of dilution factor, aeration, and agitation on Day 6 of fermentation. For fractional factorial design, two dilution factors were combined with minimum and maximum aeration and agitation rates. To check the day-to-day activity, one-way ANOVA and Tukey comparisons were performed for each treatment. Activity levels on Day 6 were considered to represent the effect of aeration and agitation. The highest enzyme activity for each treatment with respect to the day was also determined. All statistical analyses were performed using the Minitab Statistical Software (Version 19, Minitab Inc, State College, PA).

Results and Discussion

This study deals with the effect of dilution factor, aeration and agitation on the cellulase and hemicellulase activities in the fermentation media based on distillers dried grains with solubles (DDGS). The results of different activity levels on a 9-day fermentation period are given below along with the sugar consumption trends.

CELLULASE PRODUCTION TRENDS AT DIFFERENT DILUTION, AERATION, AND AGITATION RATES

Figure 1 shows the cellulase activity trends along with the sugar concentration from Day 1 to Day 9 for the combinations of factors used in this study. The enzyme production showed almost a stable increase over the entire period of 9-day incubation in case of higher dilution and lower aeration and agitation. On Day 3 and 4, there was a slight decrease, which could be due to recovering from the adaptation of the lag phase to the exponential phase (or log phase) of fungal strains for this run. The sugar concentration in the media decreased gradually over time from 11.15 g/L to 3.13 g/L within 9 days for this run as well. Maximum cellulase activity of 0.68 IU/ml was reached on Day 8, but this was not significantly different from Day 9 activity, as confirmed by the Tukey test. These activities were obtained with the lowest possible aeration and agitation rates in the bioreactor. When low aeration and agitation rates were used, higher dilution was favorable for enzyme production.

Fig. 1.

Trends of cellulase production and sugar consumption under different fermentation conditions. (a) DF4, Ae:0 and Ag:100; (b) DF4, Ae:1 and Ag:500; (c) DF2, Ae:0 and Ag:100; and (d) DF2, Ae:1 and Ag:500.

The one-way ANOVA results for different dilution factors and minimum and maximum aeration and agitation rates are given in Table 3 for cellulase activities. Tukey comparisons between days of fermentation for DF 2 with minimum aeration and agitation show similar cellulase activities except Day 3 although the highest cellulase activity was obtained on Day 6. Similarly for DF 4, Day 8 showed the highest cellulase activity and it was significantly different from only Day 1. For DF 4 with maximum aeration and agitation, the means of all days were not significantly different from each other. For DF 2, the highest cellulase activity was observed on Day 7. Therefore, the highest activity for the three treatments under consideration was achieved in the later days of fermentation.

Table 3.

ANOVA Results for Day vs. Dilution Factor with Each Combination of Aeration and Agitation

ANALYSIS OF VARIANCE FOR DAY VS DF 2 WITH 0 VVM AERATION AND 100 RPM AGITATION
Source	DF	Adj SS	Adj MS	F-Value	P-Value
Day for DF	8	0.2131	0.02663	2.52	0.049
Error	18	0.1903	0.01057
Total	26	0.4034

ANALYSIS OF VARIANCE FOR DAY VS DF 4 WITH 0 VVM AERATION AND 100 RPM AGITATION
Source	DF	Adj SS	Adj MS	F-Value	P-Value
Day for DF	8	0.2046	0.025577	3.34	0.016
Error	18	0.1377	0.007651
Total	26	0.3423

ANALYSIS OF VARIANCE FOR DAY VS DF 2 WITH 1 VVM AERATION AND 500 RPM AGITATION
Source	DF	Adj SS	Adj MS	F-Value	P-Value
Day for DF	8	0.2933	0.036666	3.97	0.007
Error	18	0.1663	0.009238
Total	26	0.4596

ANALYSIS OF VARIANCE FOR DAY VS DF 4 WITH 1 VVM AERATION AND 500 RPM AGITATION
Source	DF	Adj SS	Adj MS	F-Value	P-Value
Day for DF	8	0.04974	0.006218	0.66	0.717
Error	18	0.16886	0.009381
Total	26	0.21861

Furthermore, when the aeration and agitation rates were increased from 0 to 1 vvm and from 100 to 500 rpm respectively for DF4, no significant increase in cellulase activity was observed as shown in Fig. 1.

As shown in Fig. 1, the maximum cellulase activity was observed to be 0.61 IU/mL, which was not significantly different from 0.68 IU/mL (p > 0.05). However, one prominent aspect to notice is the sugar concentration, which decreased sharply to 0.95 g/L on Day 2. Therefore, it appears that aeration and agitation dramatically accelerate sugar consumption and cellulase production.

There have been several research studies, where the effects of aeration and agitation on various fungal enzyme activities were studied with respect to other parameters including sugar consumption. In the study of Germec and Turhan,¹³ non-aerated cultures were reported to show lower sugar consumption rates and lower biomass production with fungal inulinase activities. However, once the aeration was increased to 1 vvm, similar to this study, the enzyme production and sugar consumption rates were increased (1825.38 U/mL). ¹³ When the agitation speed was increased from 200 to 600 rpm, maximum enzyme production was obtained within 10 days. Regarding the lower agitation speeds, there are many other studies reporting enhanced enzyme production under less than 300 rpm agitation.¹⁴

Sirohi et al.¹⁴ also reported 0.372 U/mL of cellulase activity at 120 rpm agitation between Day 3 and 4 of fermentation, which was very similar to the results obtained in this study. The relationship between agitation speeds and enzyme activities was also illustrated in the study of Ahamed and Vermette,¹⁵ in which a constant agitation of 250 rpm was used and 7.1 U/mL of filter paper activity was obtained. Higher cellulolytic enzyme activities were reported at low or no agitation.¹⁶

When the fermentation media are diluted by 4X, the enzyme activities are not significantly changed by the aeration and agitation rates (Table 4). However, if the dilution is decreased by 2-fold (DF = 2), as shown in Fig. 1, higher cellulase activities are obtained especially in the later days of fermentation (Day 9).

Table 4.

ANOVA Table for the Effect of Dilution Factor (DF), and Aeration/Agitation (Ae/Ag) on Cellulase Production on Day 6 of Fermentation

SOURCE	DF	ADJ SS	ADJ MS	F-VALUE	P-VALUE
Model	3	0.065500	0.021833	26.46	0.004
Linear	2	0.020500	0.010250	12.42	0.019
DF	1	0.018050	0.018050	21.88	0.009
Ae/Ag	1	0.002450	0.002450	2.97	0.160
2-Way Interactions	1	0.045000	0.045000	54.55	0.002
DF^*Ae/Ag	1	0.045000	0.045000	54.55	0.002
Error	4	0.003300	0.000825
Total	7	0.068800

Cellulase activities with 0 vvm aeration and 100 rpm agitation rates produced significantly (p < 0.05) lower cellulase activities (0.40 IU/mL) than the cellulase activities at 1 vvm and 500 rpm (0.76 IU/mL). The sugar concentration for DF = 2 with no aeration and 100 rpm agitation remained almost constant within 9 days.

The initial sugar concentration on Day 1 was 23.11 g/L, which only decreased to 22.17 g/L on Day 9. The low sugar consumption is associated with low agitation and aeration rates, as in many other studies reporting lower nutrient consumption rates at lower agitation and aeration.¹³ The higher aeration and agitation rates with higher initial sugar concentrations showed a sharp decrease in the sugar concentrations from Day 1 (21.64 g/L) to Day 4 (1.5 g/L).

The varying trends observed in different combinations of aeration and agitation with medium dilution confirm the variability of fungal cellulolytic enzyme production under different culture conditions. Fungal cellulases are a set of cellulase enzymes including endocellulases (endoglucanase), exocellulases (exoglucanase or cellobiohydrolase), and beta-glucosidases (cellobiase).¹⁷

There are many studies, which report controversial results such that while a higher agitation rate might be beneficial to one type of cellulolytic enzyme (β-glucosidase), it might have a negative effect on another type of cellulase (endoglucanase). Studies reported by Buffo et al.^18,19 are two good examples in this regard. They also indicated that shear stress has a positive effect on β-glucosidase, while it has a negative impact on the production of endoglucanase. In this study, cellulases were measured as international units with filter paper as the substrate in the analysis. Therefore, cellulase in IU/mL represents the total activity of cellulases releasing glucose from cellulosic filter paper.

XYLANASE PRODUCTION TRENDS AT DIFFERENT DILUTION, AERATION, AND AGITATION RATES

Daily trends of xylanase activities and sugar concentration levels under varying aeration and agitation rates are shown in Fig. 2. Similar to the cellulase activities, xylanase activity increased from Day 1 to Day 9 in all tested conditions. The sugar concentration also decreased each day in all media. Maximum xylanase activity was observed to be 18.30 IU/mL on Day 9 at minimal aeration and agitation rates. The sugar concentration decreased from 11.15 to 3.13 g/L in this range of time. On the other hand, one of the highest xylanase activities was observed only on the 2^nd day of fermentation after increasing the aeration to 1 vvm and agitation to 500 rpm at the same dilution (Fig. 2b).

Fig. 2.

Trends of xylanase production and sugar consumption under different fermentation conditions. (a) DF4, Ae:0 and Ag:100; (b) DF4, Ae:1 and Ag:500; (c) DF2, Ae:0 and Ag:100; and (d) DF2, Ae:1 and Ag:500.

This xylanase activity then remained significantly unchanged through Day 9. The sugar concentration also decreased dramatically from 6.63 g/L on Day 1 to 0.95 g/L on Day 2. The aeration and agitation rates definitely influenced the time of maximum xylanase production, which was Day 9 for lower aeration and agitation rates and Day 2 for the highest aeration and agitation rates. With the help of two-way ANOVA, it was determined that xylanase activity on Day 2 at higher aeration and agitation rates was significantly higher than the xylanase activity on Day 9 with lower aeration and agitation rates for DF 2.

The effect of aeration and agitation on xylanase production has been studied extensively in the last two decades. Most of these studies corroborate the positive effect of aeration and agitation on xylanase production. One study reported that maximum xylanase production with Aspergillus niger SS7 was obtained at 1 vvm aeration.²⁰ These results are very similar to the results shown in this study. The agitation speed was 200 rpm as compared to other tested speeds such as 100 and 300. In another study, fungal species (Aspergillus nidulans) were shown to be positively affected by higher agitation rates for both sugar consumption and xylanase production.²¹ The influence of higher aeration and agitation rates, however, can be associated with other fermentation parameters such as medium composition and other process parameters like pH and temperature.^22
–24

Among various process parameters, the concentration of sugars is one of the most critical ones as not only supporting biomass growth but also leading to the production of desirable metabolites.^25,26 When the concentration of reducing sugars is increased in the media, biomass growth also increases as the fungal strains do not need to break down the complex media elements such as cellulose or hemicellulose into simple sugars.

However, it is important to determine what the optimum concentration is for enzyme production. With a dilution factor of 2 and lower aeration and agitation rates, xylanase production gradually increased until Day 6 of the fermentation, after which, the change in enzyme activity was not significant (Fig. 2c). The concentration of sugars also remained similar throughout the fermentation with no significant change. This shows a very similar trend to that of a dilution factor of 4. The maximum enzyme activity was only 13.45 IU/mL (Fig. 2). On the other hand when the aeration and agitation rates were increased to 1 vvm and 500 rpm, respectively, the maximum xylanase activity was observed to be 26.87 IU/mL on Day 3 (Fig. 2d).

The results show a positive effect of higher aeration and agitation rates as indicated by many other studies. The sharp decrease in the sugar concentration in the first three days is also correlated with this increase. However, the enzyme activity did not further increase after Day 3 and remained similar throughout the rest of the fermentation days. So, it can be concluded that with 1 vvm aeration and 500 rpm, the fermentation cycle with the given media composition can be stopped after 3 days which will decrease the overall cost of the batch fermentation cycles. Increasing aeration and agitation rates have been shown to have a positive effect on xylanase production in many studies.²¹ Therefore, it can be concluded that obtaining 26.87 IU/ml on Day 3 with higher aeration and agitation rates is significantly better than 13.45 IU/mL on Day 8, as supported by one-way ANOVA.

The overall results indicate that dilution has a significant effect on cellulase production, and its interaction with the aeration and agitation rates was also significant. On the other hand, for xylanase production, only aeration and agitation rates were correlated (R² = 0.8124) while the dilution factor and its interaction with the aeration and agitation were not significant (Table 5).

Table 5.

ANOVA Table for the Effect of Dilution Factor (DF), and Aeration/Agitation (Ae/Ag) on Xylanase Production on Day 6 of Fermentation

SOURCE	DF	ADJ SS	ADJ MS	F-VALUE	P-VALUE
Model	3	276.337	92.112	5.77	0.062
Linear	2	258.696	129.348	8.11	0.039
DF	1	6.020	6.020	0.38	0.572
Ae/Ag	1	252.675	252.675	15.84	0.016
2-Way Interactions	1	17.642	17.642	1.11	0.352
DF^*Ae/Ag	1	17.642	17.642	1.11	0.352
Error	4	63.804	15.951
Total	7	340.142

One reason for the lack of measurable effect of aeration and agitation could be due to varying responses of cellulase components to aeration and agitation rates, as reported by other various studies.^18,19 For xylanase production, on the other hand, aeration and agitation rates have a strong correlation with enzyme production. This has also been shown in many other research articles.^20,21

Conclusion

In this study, the individual effects of DDGS medium dilution, aeration, and agitation on cellulase and xylanase productions along with sugar consumption were evaluated by a fractional factorial model. The highest cellulase production (0.76 IU/mL) was obtained on Day 7 with medium dilution factor 2, aeration 1 vvm, and agitation 500 rpm. Similarly, the highest xylanase activity was 29.04 IU/mL on Day 8 with medium dilution factor 2, aeration 1 vvm, and agitation 500 rpm. Lower nutrient dilution with higher aeration and agitation rates results in higher cellulase and xylanase activities in the earlier days of fermentation. Future work should focus on the implementation of dilution factor, aeration, and agitation to obtain maximal enzyme production at a larger scale using the results of this study as a baseline.

Footnotes

Acknowledgments

The authors gratefully acknowledge Pennsylvania Grain Processing, LLC^® (Clearfield, PA, USA) for providing DDGS used in the study.

Author Contributions

Attia Iram: Conceptualization, Methodology, Data curation, Writing- Original draft preparation. Ali Demirci: Conceptualization, Visualization, Investigation, Supervision, Writing- Reviewing and Editing. Deniz Cekmecelioglu: Software, Visualization, Investigation, Writing- Reviewing and Editing, Supervision.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported in part by FULBRIGHT Student Program by providing scholarship to Attia Iram and by the USDA National Institute of Food and Agriculture Federal Appropriations under Project PEN04671 and Accession number 1017582.

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