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Article

Repeated-Batch Ethanol Fermentation from Sweet Sorghum Stem Juice under a Very High Gravity Condition Using a Stirred Tank Bioreactor Coupled with a Column Bioreactor by Immobilized Saccharomyces cerevisiae

by
Benjaporn Sriputorn
1,
Lakkana Laopaiboon
2,3 and
Pattana Laopaiboon
2,4,*
1
Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand
2
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand
3
Fermentation Research Center for Value-Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand
4
Center for Alternative Energy Research and Development, Khon Kaen University, Khon Kaen 40002, Thailand
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(2), 159; https://doi.org/10.3390/fermentation9020159
Submission received: 27 December 2022 / Revised: 3 February 2023 / Accepted: 3 February 2023 / Published: 7 February 2023
(This article belongs to the Section Fermentation Process Design)
Browse Figures
Versions Notes

Abstract

:
The ethanol fermentation efficiency of sweet sorghum stem juice (SSJ) under a very high gravity (VHG) condition (250 g/L of sugar) was improved by immobilized Saccharomyces cerevisiae SSJKKU01, using a stirred tank bioreactor (STR) coupled with a column bioreactor (CR). Dried rattan pieces (as carriers for cell immobilization) at 50% of the working volume of the CR were suitable for use in a batch ethanol fermentation. The average ethanol concentration (PE) and ethanol productivity (QP) of repeated-batch fermentation in the CR for eight successive cycles were 109.85 g/L and 1.88 g/L⋅h, respectively. Then an STR coupled with a CR was applied for repeated-batch ethanol fermentation in two systems. System I was an STR (1.8 L working volume), and System II was an STR (1 L) coupled with a CR, referred to as a CR-F (0.8 L). Both systems were connected to a new CR, called CR-I, containing sterile dried rattan pieces at 50% of its working volume. Active yeast cells were inoculated only into the STR, and the medium circulation rate between bioreactors was 5.2 mL/min. The results showed that at least eight successive cycles could be operated with an average PE of 108.51 g/L for System I and 109.44 g/L for System II. The average QP and SC values of both systems were also similar, with values of 1.87 to 1.88 g/L⋅h and 93 to 94%, respectively. The morphology of the carriers with and without immobilized cells before and after the fermentation was investigated. The obtained results demonstrated that a repeated-batch fermentation by immobilized cells on rattan pieces, using an STR coupled with a CR, was successfully used to produce high levels of ethanol from SSJ under a VHG condition.

1. Introduction

Today, the demand of fossil fuels has very much increased, so ethanol as an alternative fuel is of great interest. Normally, ethanol is produced from biomass-based fermentations, which lead to less toxic effluents and a product that is easy to integrate with transport fuel, i.e., E10 or E20, since it can be blended and used without engine modifications. The yeast Saccharomyces cerevisiae is commonly used for ethanol fermentation from several agricultural feedstocks, i.e., starch, sugars, and lignocellulosic materials [1,2].
Sweet sorghum is an interesting alternative sugar crop for ethanol production because the juice from its stalk contains high levels of fermentable sugars (sucrose, fructose, and glucose), which can be directly fermented to ethanol. Additionally, sweet sorghum stem juice (SSJ) contains many essential trace elements for yeast growth and ethanol production [3]. This crop is drought resistant and can be cultivated at nearly all temperatures. In tropical climates, it has a shorter growing period than sugarcane [4].
Our previous study successfully improved the ethanol fermentation from SSJ under a high gravity (HG) condition, containing an initial sugar concentration of 208 g/L [5]. One method to further enhance the ethanol fermentation efficiency in terms of both final ethanol titer and fermentation rate is by very high gravity (VHG) fermentation technology. A VHG fermentation involves the preparation of media containing at least 250 g/L of dissolved solids and produces ethanol at concentrations at least 15% (v/v) [6,7]. Normally, ethanol fermentations under VHG conditions are rarely completely fermented. This may be due to increased osmotic pressure, which has an adverse effect on microbial cells [6,8]. However, appropriate nutrients and process parameters, i.e., a nitrogen source, temperature, aeration, and yeast dose, can prevent the loss of cell viability, while increasing the growth rate and ethanol production under a VHG fermentation [5,9,10].
Batch ethanol fermentation is the traditional process used since it is simple to operate, as the fermentation medium and the microorganisms (yeast) are added at the beginning of the fermentation. However, this process requires much upstream work, such as inoculum and bioreactor preparation. Additionally, the yeast may be affected by substrate and product inhibition, resulting in both decreased growth and product formation. Repeated-batch fermentation is a process in which a portion of fermented medium is withdrawn at specific time intervals, and the residual medium is used as an inoculum for the next cycle. This process offers many benefits over batch fermentation. It not only reuses microbial cells for a subsequent cycle but also requires less time for the operation, with increased productivity [5,11,12].
Ethanol fermentation by free cells is a traditional process. Fermentation by immobilized cells is of interest because it has many advantages over the use of free-cell systems. The main advantages of cell immobilization are higher cell concentrations and recycling. This utilizes the cells since their separation from the medium is easier. Moreover, a carrier used for the cell immobilization effectively protects the cells, resulting in lower substrate and product inhibition [13]. Therefore, it is appropriate for use in VHG fermentations. However, the drawback of cell immobilization is a mass transfer limitation. Additionally, some carriers or supporters used for cell immobilization are expensive and unsuitable for industrial applications [14,15]. Some agriculture materials are potentially low-cost and highly available carriers that can be used for cell immobilization at an industrial scale. There are several studies on ethanol production by immobilized cells using natural carriers, e.g., corncobs [16], sorghum bagasse [17], sugarcane pieces [18], sweet sorghum stalks [19], and water hyacinth [20]. Composites of natural materials with synthetic materials such as oak chips with cellulose powder [21], Mucuna urens with glutaraldehyde [22], and alkali treated corncobs [16] were also adapted for use as carriers for cell immobilization. However, cost-effectiveness is the first important consideration. In this study, “rattan” was used as a carrier because it is highly porous and suitable for cell adsorption. Additionally, its surface is hard, thus making it durable for long-term operations. Currently, there is no report of using rattan as a carrier for cell immobilization. In Thailand, rattan has been used to make furniture and baskets, resulting in much waste from the manufacture of these items. Rattan has a potential for use as an effective carrier with no pretreatment required.
Typically, stirred tank bioreactors (STRs) have long been used for ethanol production, both at the laboratory and industry scale. The main advantage of using an STR is due to its good mixing performance. However, product inhibition can occur because of high product concentrations inside the STR [23]. Additionally, an STR presents limitations such as complexity and costly construction, mainly due to the requirement to turn an agitator shaft [24]. It was reported that a column bioreactor (CR) could be used to reduce product inhibition occurring in an STR as the product concentration in a CR increases gradually along its axial direction [24,25]. Additionally, lower power requirements are also one of the benefits of using a CR [26]. To increase the efficiency of ethanol production, many process improvements have been studied, including a fermentation system using an STR coupled in series with a CR [23,27], which can reduce product inhibition to some extent [13].
Therefore, the aim of this study was to improve the ethanol fermentation efficiency under a VHG condition from sweet sorghum juice (SSJ), using an STR coupled with a CR in a repeated-batch fermentation by immobilized yeast cells on rattan pieces. Ethanol fermentation by free cells under the same operating conditions was performed for the purpose of comparison. Furthermore, 3D digital microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and Brunauer–Emmett–Teller (BET) measurements were used to confirm the results.

2. Materials and Methods

2.1. Raw Material and Ethanol Production Medium Preparation

Sweet sorghum stalks (cv. KKU40) were obtained from the Division of Agronomy, Faculty of Agriculture, Khon Kaen University, Thailand. The juice extracted from these stalks contained ~17 °Brix of total soluble solids, which was concentrated to ~65 °Brix and stored at −20 °C until use.
An ethanol production (EP) medium was prepared by diluting the concentrated SSJ with distilled water to obtain a total sugar concentration of 250 g/L. Urea at 3 g/L was supplemented [28] into the medium before it was sterilized at 110 °C for 28 min.

2.2. Microorganism and Inoculum Preparation

S. cerevisiae SSJ01KKU [28] was inoculated into a 250 mL Erlenmeyer flask containing 150 mL of yeast extract malt extract (YM) medium containing 10 g/L of glucose (YM10), called Starter 1. After incubation on a rotating shaker at 200 rpm at 32 °C for 18 h, the culture was transferred into YM medium containing 20 g/L of glucose (YM20), called Starter 2 (modified from [19]) and incubated for another 15 h before use as an inoculum for cell immobilization. Except for the experiment in Section 2.4.3, Starter 2 was prepared with sterile SSJ containing 100 g/L of total sugar called SSJ100 (modified from [19]) and used as an inoculum for ethanol fermentation by free cells.

2.3. Carrier Preparation and Characterization

The carrier used in this study was dried rattan with diameters ranging from 4 to 6 mm and a thickness of 6 mm. It was cut into pieces by using a sharp knife (modified from [19]). The rattan was washed with tap water, followed by drying at 90 °C for 24 h or until constant weight. Dried rattan pieces were autoclaved at 121 °C for 15 min before use as carriers for cell immobilization. The characteristics of dried rattan pieces were observed by using 3D digital microscopy (HIROX KH-8700: Hackensack, NJ, USA) and scanning electron microscopy or SEM (HitachiS-3000N: Tokyo, Japan). Dried rattan surfaces were analyzed by using a Brunauer–Emmett–Teller (BET) method (Bel Sorp mini II: Osaka, Japan).

2.4. Experiments

2.4.1. Effects of Rattan Loading for Use as Carriers in Batch Ethanol Fermentation

The column bioreactor (CR) used in this study was a double-jacketed glass column with a 7 cm inner diameter and 43 cm height. Sterile rattan pieces at 30, 50, and 70% of its working volume (corresponding to 22, 37, and 51 g of rattan pieces) were packed into the CR. Then sterile SSJ100 medium (350 mL) (from Section 2.1) was added into the CR. An inoculum containing 1 × 108 cells/mL of yeast cells from Starter 2 (from Section 2.2) was added into the CR. The immobilization process was carried out at 32 °C for 24 h. After that, the carriers with immobilized cells were washed with fresh SSJ100 medium. Then EP medium (350 mL) was added into the CR to start the ethanol fermentation at 32 °C.

2.4.2. Repeated-Batch Ethanol Fermentation Using a CR

Repeated-batch fermentation under the optimum rattan loading (from Section 2.4.1) was first carried out in batch mode (Section 2.4.1) until the sugar concentration in the medium remained approximately 10% of its initial value. Then all the fermented medium was withdrawn, leaving only the immobilized cells on the carriers in the CR. After that, the same amount of the fresh EP medium was added to start the next cycle. The repeated-batch fermentations were performed for eight successive cycles or until the fermentation efficiency was lower than 25% of the first cycle. The repeated-batch fermentation was controlled at 32 °C, and sterile air was simultaneously supplied at 2.5 vvm for 4 h at the beginning of each cycle of repeated-batch fermentation. Additionally, air was supplied at the bottom of each column at 0.005 vvm to prevent cell settling during fermentation [28]. A schematic diagram and images of the system for repeated-batch fermentation using a CR are shown in Figure 1A-1 and A-2, respectively.

2.4.3. Repeated-Batch Ethanol Fermentation Using STR Coupled with CR

The STR (Biostat® M, B. Braun Biotech, Melsungen, Germany) coupled with CR(s) was used in repeated-batch fermentation as two systems. System I comprised an STR containing 1.8 L of sterile EP medium, while System II was an STR (containing 1 L of EP medium) coupled with CR (CR-F) (containing 0.8 L of EP medium). These two systems were connected to a new CR (CR-I) containing sterile dried rattan pieces (as carriers for cell immobilization) at the desired rattan loading (from Section 2.4.1). The inoculum from Starter 2 (Section 2.2) was added only into the STR to obtain an initial cell concentration in the STR of 5 × 107 cells/mL. After that, sterile EP medium was immediately circulated between the STR and CR-I (for System I) or STR, CR-F, and CR-I (for System II) at a flow rate of 5.2 mL/min [28]. Sterile air was simultaneously supplied at 2.5 vvm for 4 h at the beginning of each cycle of repeated-batch fermentation in the STR, and it was supplied at the bottom of each column at 0.005 vvm to prevent cell settling during fermentation [28]. The fermentation was carried out until the sugar concentration in the medium reached approximately 10% of its initial value. Then all of the fermented medium was withdrawn from all bioreactors. After that, the same amount of sterile EP medium was added into the STR of System I or into the STR and CR-F of System II to start the next cycle. The repeated-batch fermentations were performed for at least eight successive cycles or until the fermentation efficiency was lower than 25% of the first cycle. The fermentation temperature was controlled at 32 °C. A schematic diagram of the System I and System II used in repeated-batch fermentation and their images are shown in Figure 1B-1,B-2,C-1,C-2, respectively.

2.5. Analytical Methods

During all fermentations, samples were collected at 12 h intervals for analyses. The free viable yeast cell numbers in the fermentation medium were determined with a direct counting method, using a haemocytometer with methylene blue staining. The pH and total soluble solids of the fermentation broth were determined directly, using a pH meter and handheld refractometer, respectively [29]. At the end of the fermentation, the appearance of the immobilized cells on rattan pieces was observed using 3D digital microscopy (HIROX KH-8700: USA). The rattan pieces were coated with a 2 μm thick layer of gold under a 1 mbar pressure for 15 min before being observed using scanning electron microscopy (SEM, HitachiS-3000N: Japan) [30,31]. Additionally, the surface topography of rattan pieces was imaged using atomic force microscopy (AFM, XE-120, Park Systems, Republic of Korea). The specific surface area, pore volume, and mean pore diameter of rattan pieces were determined by using a Brunauer–Emmett–Teller (BET) method.
The fermentation broth was centrifuged at 12,000 rpm for 10 min to remove yeast cells and solid particles, and the supernatant was analyzed for total sugar and ethanol concentration. The total sugar concentration was measured using a phenol–sulfuric acid method [32]. Ethanol concentration was analyzed using a gas chromatograph with a flame ionization detector employing propanol as an internal standard. The stationary phase was a polyethylene glycol packed column (PEG-20 M). The column and injection temperatures were controlled at 150 and 180 °C, respectively. The temperature of the detector was 250 °C. Nitrogen gas was used as a carrier [5]. Ethanol productivity (QP, g/L⋅h) was calculated as the ethanol concentration produced (PE, g/L) divided by fermentation time (t). Percentage of sugar consumption (SC, %) was calculated as the actual total sugar utilized divided by the initial total sugar concentration multiplied by 100. Ethanol yield (YP/S) was calculated as the PE produced (g/L) divided by the total sugar utilized (g/L).

3. Results and Discussion

3.1. Appearance and Morphology of the Rattan

The selection of a carrier is a crucial decision that is made during the development of an immobilization process. It needs to be a non-toxic carrier with a simple immobilization procedure, have high biomass retention, and be preferably a low cost [33]. Therefore, the physical characteristics of dried rattan pieces that we decided to use as the carriers in the current study were first observed. The appearance of the rattan pieces under 3D digital microscopy revealed much porosity distributed on the surface, as shown in Figure 2.
When the ultra-structure of dried rattan pieces was analyzed using SEM, the results showed that the rattan structure and its surface were rough. The rattan had many cavities in both the top and cross-sectional views (Figure 3). These structures are advantageous for cell immobilization because they allow microorganisms to attach more firmly than for smooth structures [34]. Therefore, dried rattan pieces have high potential for use as low-cost carriers for cell immobilization with no pretreatment.
Additionally, the dried rattan was analyzed using a BET method to determine the surface-area properties of the rattan since this greatly influenced the cell adsorption [33,35]. The dried rattan had a specific surface area of 0.292 m2/g and a pore volume of 0.0058 cm3/g. María et al. [36] reported that the specific surface area of wood shavings, corn leaves, bagasse, and corncobs ranged from 2.2 to 5.3 m2/g, which is markedly higher than that of dried rattan. However, the pore volume of dried rattan (0.0058 cm3/g) was in the range of other lignocellulosic materials (0.004 to 0.006 cm3/g). To the best of our knowledge, no report is available on using dried rattan as a carrier for cell immobilization. Therefore, this report will be helpful for the utilization of dried rattan as a potential low-cost carrier for cell immobilization in ethanol fermentation.

3.2. Batch Ethanol Fermentation by Immobilized Cells on Rattan Pieces at Various Carrier Loadings in a CR

The profiles of viable cells, sugar, and ethanol concentrations during batch ethanol fermentation by immobilized cells at various loadings of dried rattan in a CR are shown in Figure 4. The initial free-cell concentrations in the EP medium were 4.38 × 106, 2.23 × 106, and 3.63 × 106 cells/mL, using 30, 50, and 70% of the CR volume for dried rattan pieces, respectively. The initial sugar concentrations were 250.59, 245.92, and 243.85 g/L, using 30, 50, and 70% of the CR for dried rattan pieces, respectively. The total sugar concentration decreased to approximately 22 g/L in all conditions at the end of fermentation. At 54 h of fermentation time, the PE values at all conditions were not significantly different, ranging from 106.15 to 108.70 g/L, corresponding to the SC values of 90 to 93% and QP values of approximately 2 g/L⋅h (Table 1). The ethanol yield (YP/S) under all conditions tested was not different, suggesting that cell immobilization on rattan pieces did not affect the metabolic pathway of ethanol fermentation. The results indicate that the dried rattan pieces were successfully used as carriers for cell immobilization to produce ethanol, and varying carrier loadings from 30 to 70% had no marked effect on ethanol production. However, to prevent any damage to the carriers under long-term operation in repeated-batch fermentation, 50% of dried rattan was selected for subsequent experiments.

3.3. Repeated-Batch Fermentation in a CR by Immobilized Cells

Repeated-batch fermentations were conducted for eight successive cycles, with a total fermentation time of 468 h (Figure 5). The initial free-cell concentration in the EP medium was 8.15 × 106 cells/mL in Cycle 1, and it was 1.70 to 3.75 × 107 cells/mL in Cycles 2 to 8, indicating that the immobilized yeast cells of the previous cycle had detached and could grow well in the broth. The initial pH values in all cycles were 4.78 ± 0.04. Changes of pH during the fermentation in Cycles 1 to 8 were similar, with values of 4.08 to 4.61 (Figure 5A). In the first cycle, the initial sugar concentration was 251.71 g/L and decreased to 17.22 g/L at 54 h of fermentation time. The average initial sugar concentrations of Cycles 2 to 8 were 251.47 ± 1.81 g/L. The fermentation time of Cycle 2 was also 54 h, with 23.71 g/L sugar remaining. Due to a higher level of residual sugar at the end of Cycle 2 compared to Cycle 1, the fermentation time of cycles in Cycles 3 to 8 was extended to 60 h. However, the residual sugar concentrations in Cycles 3 to 8 slightly increased (26.08 to 29.54 g/L) compared to that in Cycle 2. The average SC of eight successive cycles was 89.59 ± 1.64%. The PE values of each cycle ranged from 106.56 to 115.27 g/L, with an average PE value of 109.85 ± 3.17 g/L (Figure 5B). Similar PE values in the repeated-batch fermentation by the immobilized cells on rattan pieces indicated that more than eight cycles could be operated by this system. The total working volume in the eight cycles was 2.8 L (350 mL/cycle) in 468 h. Thus, the total amount of ethanol produced was 308 g, corresponding to an ethanol production rate of 0.66 g/h. To increase the ethanol production rate, ethanol production at a higher total working volume, using combined bioreactors comprising an STR coupled with a CR, was used in the subsequent experiments.

3.4. Repeated-Batch Ethanol Fermentation by Immobilized Cells Using an STR Coupled with a CR

Repeated-batch fermentations with two systems, System I and System II (Section 2.4.3), were carried out.

3.4.1. System I (STR Coupled with CR-I)

A repeated-batch fermentation was performed for eight successive cycles, using System I, an STR (1.8 L of working volume) connected to CR-I containing immobilized cells as inoculum of each cycle. In the first cycle, the fermentation time was 48 h, and it was prolonged to 60 h in Cycles 2 to 8, for a total fermentation time of 468 h. The average initial pH of the medium in each cycle was 4.53 ± 0.07, and the pH changes of the medium in the STR and CR-I in all cycles were similar, ranging from 3.90 to 4.52 (data not shown). In each cycle, the changes of viable yeast cell counts (Figure 6A), total sugar, and ethanol concentration (Figure 6B) were similar to those in the repeated-batch fermentation using only a CR (Figure 5). The initial free-cell concentration in the first cycle in STR was 4.88 × 107 cells/mL, and it ranged from 0.8 to 2.68 × 107 cells/mL in Cycles 2 to 8. During the fermentation, the viable cell counts in CR-I were slightly lower than those in the STR. This might have been due to better mixing in the STR. At the end of each cycle, the cell numbers were relatively high at 1.62 to 2.40 × 108 and 1.34 to 2.15 × 108 cells/mL in the STR and CR-I, respectively (Figure 6A). It was found that non-viable cells in Cycle 1 were lowest, and the values increased, ranging from 4.25 × 106 to 1.38 × 107 cells/mL, in Cycles 2 to 8 (Figure 6A), suggesting that non-viable cells in rattan pieces as carriers from previous cycles might have detached. The profiles of sugar consumption and ethanol production of Cycles 1 to 8 were quite similar (Figure 6B). An average initial sugar concentration, 252.91 ± 0.91 g/L, was observed with an average final sugar concentration of 15.62 ± 3.49 g/L. The PE values in the STR and CR-I were not different, with average values of 108.51 ± 1.98 g/L. In System I, the total working volume in the eight successive cycles was 14.4 L (1.8 L/cycle), and the total amount of ethanol produced was 1563 g, corresponding to an ethanol production rate of 3.34 g/h (Table 2). Additionally, a high sugar consumption of 93.83% was observed under this condition.

3.4.2. System II (STR Coupled with CR-F and CR-I)

To reduce the capital costs of the STR, System II was operated with a smaller STR (1.0 L working volume) coupled with a CR (CR-F) (0.8 L working volume). It was connected to CR-I containing immobilized cells as an inoculum of each cycle. The total working volumes of Systems I and II were the same, 1.8 L. The results showed that at least eight successive cycles of repeated-batch fermentation could be operated using System II. The fermentation time of the first cycle was 48 h, and it was extended to 60 h in Cycles 2 to 8. Therefore, the total fermentation time was 468 h, which was equal to that of System I (Figure 7). The initial pH values of each cycle in the STR, CR-F, and CR-I were similar, with an average value of 4.39 ± 0.17. The pH changes of the medium in the STR, CR-F, and CR-I in all cycles were similar, and the pH values ranged from 3.76 to 4.61 (data not shown). The initial cell concentration in the STR in Cycle 1 was 5.55 × 107 cells/mL (Figure 7A). During the fermentation, the changes of the number of viable and non-viable cells in each cycle were similar to those reported for System I. At the end of each cycle, the viable cell concentrations ranged from 1.49 to 2.43 × 108, 1.38 to 2.33 × 108, and 1.52 to 2.38 × 108 cells/mL in the STR, CR-F, and CR-I, respectively (Figure 7A). The number of non-viable cells increased in all bioreactors during the fermentation with maximum values of 8.50 × 105 to 9.20 × 107 cells/mL (Figure 7A). The profiles of sugar consumption and ethanol production of Cycles 1 to 8 were quite similar (Figure 7B), indicating that the behavior of yeast cells in all cycles had not changed and the cells were very active. The average initial sugar concentration was 252.61 ± 3.50 g/L, with an average final sugar concentration of 18.66 ± 3.45 g/L. PE values were not different among bioreactors in each cycle, and the average PE value of eight cycles was 109.44 ± 1.50 g/L (Table 2). In System II, the total working volume for eight successive cycles was 14.4 L (1.8 L/cycle), and the total amount of ethanol produced was 1576 g, corresponding to a 3.37 g/h ethanol production rate (Table 2). These values were similar to those of System I, indicating that System II, which used an STR combined with a CR could be used instead of a system using only an STR. Additionally, the immobilized cells in the CR (CR-I) in both systems could be used as an active inoculum in repeated-batch fermentations.
Table 2 compares ethanol production of the repeated-batch fermentations by the immobilized cells, using Systems I and II, and by free cells, using an STR coupled with a CR with a 90% drain and fill volume [28]. The PE value of repeated-batch fermentation by the immobilized cells in the current study (108.51 and 109.44 g/L) was 7 to 8 g/L lower than that using free cells (116.52 g/L). However, the QP * values in terms of grams of ethanol produced per hour in this study were slightly higher than for free cells. It is projected that the differences in QP values (g/h) will be much higher when using large-scale fermenters. The PE value in the repeated-batch fermentation by the immobilized cells did not increase, while the QP * values from using both immobilized cells systems slightly increased. This occurred because all fermented broth was harvested from the immobilized cells systems, whereas some portion (10%) of the fermented broth using the free-cells system remained for use as an inoculum in the next cycle.
Apart from the decreased capital costs of the process using a CR with immobilized cells on rattan pieces, the system with immobilized cells for ethanol production enabled easy separation of the cells from the fermented broth. Liu et al. [13] reported that a repeated-batch ethanol fermentation by immobilized cells led to a higher economic benefit compared to fermentation by free cells. The immobilized cell systems produced about a 26% higher marginal value than free-cell systems because of lower fermenter, centrifuge, and filtration costs [37]. Moreover, the design of new bioreactor systems (e.g., System II, an STR coupled with CR-F and CR-I) is a challenge in commercial ethanol production.

3.5. Characteristics of Immobilized Cells on Rattan in Repeated-Batch Ethanol Fermentation

To confirm that the yeast cells were firmly immobilized on the dried rattan pieces after eight cycles of repeated-batch fermentation, the dried rattan pieces in CR-I (top and cross-sectional views) were observed under SEM. The results showed many yeast cells attached to the rattan after the end of the fermentation process (Figure 8A–F). Visual observation clearly indicated that the rattan structure was not destroyed after repeated-batch fermentation in either of the systems. Many yeast cells were still observed both in the top (Figure 8A,C,E) and cross-sectional views (Figure 8B,D,F) of rattan pieces. Cell attachment on surfaces by non-covalent interaction occurs through four mechanisms, Lifshitz theory of van der Waals forces, electrostatic forces, Lewis acid–base forces, and Brownian motion [38]. These results indicate that rattan is a highly feasible carrier for cell immobilization to produce ethanol. Additionally, the method for cell immobilization using dried rattan pieces as carriers, adsorption, is very simple. Therefore, the cost of carrier preparation and cell immobilization can be substantially reduced.
Not as expected, long-term operation in repeated-batch fermentation did not destroy the rattan structure. This may have occurred because rattan stems contain high cellulose (39–58%) and lignin (18–27%) contents [39], which directly give rattan its strength. According to Figure 4, varying carrier loading from 30 to 70% had no marked effect on batch ethanol production. Therefore, to reduce the cost of the carrier used, carrier loading at 30% or lower should be studied in repeated-batch fermentation.
The 3D structure of rattan surfaces was observed, and the roughness of the rattan was estimated by using an AFM technique; the results are shown in Figure 9A. The roughness of the rattan surface was 0.207 ± 0.01 μm, which is bumpier than a silicon surface (0.001 μm) [40] and plastic surface (0.002 nm) [41]. According to the 3D digital microscopy and SEM results (Figure 2 and Figure 3), dried rattan is very porous, resulting in high roughness. Rattan surfaces with immobilized cells after repeated-batch fermentation in System II (Figure 9B) revealed semicircular-shaped immobilized yeast cells, as seen in the SEM results. The roughness value of the rattan with immobilized cells increased to 0.731 ± 0.03 μm, confirming that many yeast cells were still attached to the rattan surface. Therefore, rattan, a widely available and low-cost agriculture material, is a potential carrier for cell immobilization to produce bioethanol.
Repeated-batch ethanol fermentation processes using immobilized S. cerevisiae from various substrates under high gravity (HG) and very high gravity (VHG) conditions are compared in Table 3. Many agricultural materials, including rattan, have been successfully used as carriers for cell immobilization. Sweet sorghum stem juice gave an ethanol yield (0.48 g/g) as high as that using glucose as a substrate. Differences in ethanol production in terms of PE, QP, SC, and YP/S are mainly due to the variations in substrate, yeast species and operating conditions.

4. Conclusions

Based on the results of this study, it can be concluded that dried rattan is a suitable low-cost carrier for cell immobilization to produce ethanol. The immobilized cells in dried rattan pieces could be used in repeated-batch ethanol fermentation for at least eight successive cycles. The CR (as a CR-I) was packed with a rattan carrier for cell immobilization. It acted as a source of inoculum for each cycle of repeated-batch fermentation. Additionally, operation with a medium circulating between the STR and CR-I (System I) or CR-F and CR-I (System II) could promote yeast growth during the fermentation process. Furthermore, a separate immobilization step was not necessarily since cell immobilization and ethanol fermentation occurred simultaneously during repeated-batch fermentation. This study demonstrates the feasibility of using combined bioreactors, e.g., an STR coupled with a CR for ethanol fermentation from SSJ under a VHG condition by immobilized cells as a cost-effective bioreactor. An STR coupled with a CR could be used for ethanol production for a higher economic benefit compared to the use of a traditional STR.

Author Contributions

Conceptualization, P.L. and L.L.; methodology and formal analysis, B.S.; investigation and writing—original draft preparation, B.S. and P.L.; writing—review and editing, P.L. and L.L.; supervision, P.L. and L.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Fundamental Fund of Khon Kaen University; the National Science, Research and Innovation Fund (NSRF), Thailand (Grant. No. 161762); and the Center for Alternative Energy Research and Development, Khon Kaen University, Khon Kaen, Thailand.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

Division of Agronomy, Faculty of Agriculture, Khon Kaen University, Thailand for providing sweet sorghum stem juice.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Fermentation 09 00159 g001 550
Figure 1. Schematic diagrams and images of ethanol fermentation using only CR (A-1,A-2), System I: STR coupled with CR-I (B-1,B-2). System II: STR coupled with CR-F and CR-I (C-1,C-2).
Figure 1. Schematic diagrams and images of ethanol fermentation using only CR (A-1,A-2), System I: STR coupled with CR-I (B-1,B-2). System II: STR coupled with CR-F and CR-I (C-1,C-2).
Fermentation 09 00159 g001
Fermentation 09 00159 g002 550
Figure 2. Images of the physical characteristics of dried rattan pieces obtained by 3D digital microscopy (A) 100× and (B) 350×.
Figure 2. Images of the physical characteristics of dried rattan pieces obtained by 3D digital microscopy (A) 100× and (B) 350×.
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Fermentation 09 00159 g003 550
Figure 3. Scanning electron microscopic images of top view (A) and cross-sectional view (B) of dried rattan pieces at a magnification of 3000×.
Figure 3. Scanning electron microscopic images of top view (A) and cross-sectional view (B) of dried rattan pieces at a magnification of 3000×.
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Fermentation 09 00159 g004 550
Figure 4. Profiles of batch ethanol fermentation in CR by immobilized cells on rattan pieces at various carrier loadings: 30% (white symbols), 50% (gray symbols), and 70% (black symbols) of dried rattan pieces (Fermentation 09 00159 i001, Fermentation 09 00159 i002, Fermentation 09 00159 i003 = viable cells; Fermentation 09 00159 i004, Fermentation 09 00159 i005, Fermentation 09 00159 i006= total sugar and Fermentation 09 00159 i007, Fermentation 09 00159 i008, Fermentation 09 00159 i009 = ethanol concentration).
Figure 4. Profiles of batch ethanol fermentation in CR by immobilized cells on rattan pieces at various carrier loadings: 30% (white symbols), 50% (gray symbols), and 70% (black symbols) of dried rattan pieces (Fermentation 09 00159 i001, Fermentation 09 00159 i002, Fermentation 09 00159 i003 = viable cells; Fermentation 09 00159 i004, Fermentation 09 00159 i005, Fermentation 09 00159 i006= total sugar and Fermentation 09 00159 i007, Fermentation 09 00159 i008, Fermentation 09 00159 i009 = ethanol concentration).
Fermentation 09 00159 g004
Fermentation 09 00159 g005 550
Figure 5. Profiles of pH ((A): Fermentation 09 00159 i010), viable cells ((A): Fermentation 09 00159 i011), total sugar ((B): Fermentation 09 00159 i012), and ethanol ((B): Fermentation 09 00159 i013) concentrations during repeated-batch ethanol fermentation by immobilized cells in a CR. The circled numbers indicate the cycle number of the fermentation.
Figure 5. Profiles of pH ((A): Fermentation 09 00159 i010), viable cells ((A): Fermentation 09 00159 i011), total sugar ((B): Fermentation 09 00159 i012), and ethanol ((B): Fermentation 09 00159 i013) concentrations during repeated-batch ethanol fermentation by immobilized cells in a CR. The circled numbers indicate the cycle number of the fermentation.
Fermentation 09 00159 g005
Fermentation 09 00159 g006 550
Figure 6. Profiles of viable cells ((A): Fermentation 09 00159 i014,Fermentation 09 00159 i015), non-viable cells ((A): Fermentation 09 00159 i016,Fermentation 09 00159 i017), sugar ((B): Fermentation 09 00159 i018,Fermentation 09 00159 i019), and ethanol ((B): Fermentation 09 00159 i020,Fermentation 09 00159 i021) concentrations during repeated-batch fermentation by immobilized cells in System I: STR (black symbols) coupled with CR-I (white symbols). The circled numbers indicate the cycle number of the repeated-batch fermentation.
Figure 6. Profiles of viable cells ((A): Fermentation 09 00159 i014,Fermentation 09 00159 i015), non-viable cells ((A): Fermentation 09 00159 i016,Fermentation 09 00159 i017), sugar ((B): Fermentation 09 00159 i018,Fermentation 09 00159 i019), and ethanol ((B): Fermentation 09 00159 i020,Fermentation 09 00159 i021) concentrations during repeated-batch fermentation by immobilized cells in System I: STR (black symbols) coupled with CR-I (white symbols). The circled numbers indicate the cycle number of the repeated-batch fermentation.
Fermentation 09 00159 g006
Fermentation 09 00159 g007 550
Figure 7. Profiles of viable cells ((A): Fermentation 09 00159 i022,Fermentation 09 00159 i023,Fermentation 09 00159 i024), non-viable cells ((A): Fermentation 09 00159 i025,Fermentation 09 00159 i026,Fermentation 09 00159 i027), sugar ((B): Fermentation 09 00159 i028,Fermentation 09 00159 i029,Fermentation 09 00159 i030), and ethanol ((B): Fermentation 09 00159 i031,Fermentation 09 00159 i032,Fermentation 09 00159 i033) concentrations during repeated-batch fermentation by immobilized cells in System II: STR (black symbols) coupled with CR-F (gray symbols) and CR-I (white symbols), respectively. The circled numbers indicate the cycle number of the repeated-batch fermentation.
Figure 7. Profiles of viable cells ((A): Fermentation 09 00159 i022,Fermentation 09 00159 i023,Fermentation 09 00159 i024), non-viable cells ((A): Fermentation 09 00159 i025,Fermentation 09 00159 i026,Fermentation 09 00159 i027), sugar ((B): Fermentation 09 00159 i028,Fermentation 09 00159 i029,Fermentation 09 00159 i030), and ethanol ((B): Fermentation 09 00159 i031,Fermentation 09 00159 i032,Fermentation 09 00159 i033) concentrations during repeated-batch fermentation by immobilized cells in System II: STR (black symbols) coupled with CR-F (gray symbols) and CR-I (white symbols), respectively. The circled numbers indicate the cycle number of the repeated-batch fermentation.
Fermentation 09 00159 g007
Fermentation 09 00159 g008 550
Figure 8. Scanning electron micrographs of immobilized yeast cells on rattan pieces after repeated-batch fermentation: top view (A,C,E) and cross-sectional view (B,D,F) of rattan after repeated-batch fermentation in a CR only (A,B), System I (C,D), and System II (E,F) at a magnification of 3000×.
Figure 8. Scanning electron micrographs of immobilized yeast cells on rattan pieces after repeated-batch fermentation: top view (A,C,E) and cross-sectional view (B,D,F) of rattan after repeated-batch fermentation in a CR only (A,B), System I (C,D), and System II (E,F) at a magnification of 3000×.
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Fermentation 09 00159 g009 550
Figure 9. Three-dimensional view from AFM imaging of dried rattan pieces (A) and rattan with immobilized cells after repeated-batch fermentation in System II (B).
Figure 9. Three-dimensional view from AFM imaging of dried rattan pieces (A) and rattan with immobilized cells after repeated-batch fermentation in System II (B).
Fermentation 09 00159 g009
Table
Table 1. Batch ethanol fermentation efficiency in a CR by immobilized cells at different rattan loadings.
Table 1. Batch ethanol fermentation efficiency in a CR by immobilized cells at different rattan loadings.
Percentage of Rattan (%)PE (g/L)QP (g/L⋅h)SC (%)YP/S (g/g)t (h)
30108.70 ± 1.91 a2.01 ± 0.04 a93.42 ± 0.06 a0.46 ± 0.01 b54
50106.15 ± 2.42 a1.97 ± 0.05 a90.35 ± 0.02 c0.48 ± 0.01 a54
70106.25 ± 0.95 a1.93 ± 0.02 a90.38 ± 0.07 b0.47 ± 0.01 ab54
PE = ethanol concentration, QP = ethanol productivity, SC = sugar consumption (%), YP/S = ethanol yield (g/g), and t = fermentation time (h). a, b, c, ab Means having the same letter within the same column are not significantly different when using Duncan’s multiple range test at a p-level < 0.05. The results are expressed as the mean ± SD of triplicate experiments.
Table
Table 2. Repeated-batch ethanol fermentation efficiency by immobilized cells in different systems.
Table 2. Repeated-batch ethanol fermentation efficiency by immobilized cells in different systems.
SystemPE (g/L)QP (g/L⋅h)Total SC (%)Total YP/S(g/g)PE * (g)t * (h)QP * (g/h)
CR109.85 ± 3.17 b1.88 ± 0.14 a89.59 ± 1.64 b0.49 ± 0.01 a---
System I (STR & CR-I)108.51 ± 1.98 b1.87 ± 0.17 a93.83 ± 1.37 a0.46 ± 0.01 b15634683.34
System II (STR & CR-F & CR-I)109.44 ± 1.50 b1.88 ± 0.16 a92.61 ± 1.34 ab0.47 ± 0.01 ab15764683.37
Free cells in STR & CR * [32]116.52 ± 2.21 a2.00 ± 0.19 a92.98 ± 0.78 a0.49 ± 0.02 a1532471.53.25
System I: repeated-batch fermentation in STR coupled with CR-I. System II: repeated-batch fermentation in STR coupled with CR-F and CR-I. * Repeated-batch fermentation by free cells in STR coupled with CR with 90% drain and fill volume. PE * = total amount of ethanol, t * = total fermentation time, and QP * = total ethanol productivity. a, b, ab Means having the same letter within the same column are not significantly different using Duncan’s multiple range test at a p-level < 0.05. The results are the mean ± SD of eight successive cycles.
Table
Table 3. Repeated-batch ethanol fermentation with immobilized S. cerevisiae on various carriers.
Table 3. Repeated-batch ethanol fermentation with immobilized S. cerevisiae on various carriers.
SubstrateInitial Sugar (g/L)S. cerevisiaeCarrierCyclesPE (g/L)QP (g/L⋅h)SC (%)YP/S (g/g)
Sugarcane molasses [42]231SC90Delignified sugarcane molasses5---0.34–0.42
Blackstrap molasses [43]240M30Thin-shell silk cocoons577.6–88.31.62–1.8486.11–92.890.44–0.48
Glucose [44]2001308Fibrous matrix2291.5711.751000.46
Glucose and sucrose [45]2803013Sweet sorghum pith10130.122.0098.210.48
Sweet sorghum stem juice (from this study, System II)250SSJ01Rattan8109.441.8892.610.47
PE = ethanol concentration, QP = ethanol productivity, SC = sugar consumption (%), YP/S = ethanol yield (g/g). System II: repeated-batch fermentation in STR coupled with CR-F and CR-I.
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Sriputorn, B.; Laopaiboon, L.; Laopaiboon, P. Repeated-Batch Ethanol Fermentation from Sweet Sorghum Stem Juice under a Very High Gravity Condition Using a Stirred Tank Bioreactor Coupled with a Column Bioreactor by Immobilized Saccharomyces cerevisiae. Fermentation 2023, 9, 159. https://doi.org/10.3390/fermentation9020159

AMA Style

Sriputorn B, Laopaiboon L, Laopaiboon P. Repeated-Batch Ethanol Fermentation from Sweet Sorghum Stem Juice under a Very High Gravity Condition Using a Stirred Tank Bioreactor Coupled with a Column Bioreactor by Immobilized Saccharomyces cerevisiae. Fermentation. 2023; 9(2):159. https://doi.org/10.3390/fermentation9020159

Chicago/Turabian Style

Sriputorn, Benjaporn, Lakkana Laopaiboon, and Pattana Laopaiboon. 2023. "Repeated-Batch Ethanol Fermentation from Sweet Sorghum Stem Juice under a Very High Gravity Condition Using a Stirred Tank Bioreactor Coupled with a Column Bioreactor by Immobilized Saccharomyces cerevisiae" Fermentation 9, no. 2: 159. https://doi.org/10.3390/fermentation9020159

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