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Article

Cell Recycling Application in Single-Stage and Sequential-Stage Co-Production of Xylitol and Ethanol Using Corn Cob Hydrolysates

by
Kritsadaporn Porninta
1,2,
Chatchadaporn Mahakuntha
2,3,
Julaluk Khemacheewakul
2,3,
Charin Techapun
2,3,
Yuthana Phimolsiripol
2,3,
Pornchai Rachtanapun
2,3,
Kittisak Jantanasakulwong
2,3,
Juan Feng
2,3,
Su Lwin Htike
2,3,
Rojarej Nunta
2,4,
Xinshu Zhuang
5,
Wen Wang
5,
Wei Qi
5,
Zhongming Wang
5,
Sumeth Sommanee
2,3,* and
Noppol Leksawasdi
2,3,*
1
Program in Biotechnology, Multidisciplinary and Interdisciplinary School, Chiang Mai University, Chiang Mai 50200, Thailand
2
Center Excellence of Agro Bio-Circular-Green (Agro BCG), School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand
3
Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand
4
Division of Food Innovation and Business, Faculty of Agricultural Technology, Lampang Rajabhat University, Lampang 52100, Thailand
5
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, CAS Key Laboratory of Renewable Energy, Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, China
*
Authors to whom correspondence should be addressed.
Agriculture 2024, 14(7), 1062; https://doi.org/10.3390/agriculture14071062
Submission received: 1 June 2024 / Revised: 27 June 2024 / Accepted: 28 June 2024 / Published: 30 June 2024
(This article belongs to the Special Issue Agricultural Waste—Status and Future Prospects)
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Abstract

:
A sustainable bioeconomy in agricultural and agro-industrial production must inevitably involve the sustainable use of agricultural residues through zero-waste processes. Corn cob is considered crucial agricultural waste as 278 and 293 million tons were produced worldwide in 2022 and 2023, respectively. Corn cob hydrolysates, which are abundant in xylose and glucose, could be efficiently utilized for xylitol and ethanol production through the cultivation of recycling the yeast strain Candida magnoliae TISTR 5664 in the single-stage and sequential-stage co-production of these products. The statistically significant maxima (p ≤ 0.05) ethanol concentrations were improved by 7.8% (49.9–51.7 g/L or 91.3–95.6% of the theoretical) from the single stage of ethanol production employing recycled cells and 9.9% (50.9–54.1 g/L or 77.3–83.9% of the theoretical) from the second step of sequential-stage co-production using recycled cells without xylitol accumulation. Conversely, the single-stage xylitol production utilizing recycled cells under microaerobic conditions resulted in a statistically significant lower (p ≤ 0.05) xylitol concentration by two folds relative to the control, while ethanol concentration was elevated by almost double. The statistically significant maximum (p ≤ 0.05) xylitol was achieved at 25.9 g/L (58.6% of the theoretical) when sequential-stage co-production was initiated in the first step with fresh inoculum only and not recycled cells. The sequential-stage co-production of xylitol and ethanol presented the potential for statistically significant improvement (p ≤ 0.05) of both xylitol and ethanol production processes.

1. Introduction

Worldwide ethanol production increased over time from 2007 to 2023, excluding the year 2020, when global production dropped due to the COVID-19 pandemic. Production has since increased (107 billion L in 2022), and it exceeded prepandemic levels for the first time in 2023 (119 billion L). The USA and Brazil contributed 80% of global ethanol production. The latter country predominantly employs sugarcane, whereas corn dominates the ethanol production in the USA [1,2]. Bioethanol could be produced from various raw materials such as sugarcane, cassava, soybean, rapeseed, and corn for first-generation biofuels. These were compared with nonedible raw materials such as Napier grass, corn cob, and sugarcane bagasse for second-generation biofuels or third-generation biofuels, such as genetically engineered algae and fast-growing poplar trees. Genetically engineered microorganisms such as cyanobacteria were employed for the fourth-generation biofuel production processes [3,4]. In fact, third- and fourth-generation biofuels are still being investigated at the pilot scale to optimize corresponding economic viability [5].
Xylitol is a low-calorie sweetener with a low glycemic index that is generally produced through the chemical process of catalytic hydrogenation. In this case, pure xylose from birch, hard wood, corn, and others is obtained prior to subsequent conversion to xylitol after specific treatments at high temperatures and pressures. Nevertheless, the drawback of this method includes significant energy consumption and the involvement of numerous reaction steps [6]. The global market of xylitol grew by 6.6% of the compound annual growth rate (CAGR) between 2022 and 2023, and it is expected to reach 6.7% CAGR in 2027 [7].
Corn cob is one of the feedstocks for the production of second-generation biofuel, which comprises relatively high cellulose and hemicellulose contents. The respective lignocellulosic contents (by weight) include 34.9 ± 0.2% cellulose, 38.3 ± 0.4% hemicellulose, and 7.80 ± 0.15% lignin [8]. After pretreatment and enzymatic hydrolysis processes, corn cob could be converted to useful monosaccharides such as glucose, xylose, and arabinose. These sugars could then be used as carbon sources to produce bioethanol and xylitol through microorganism cultivation, thereby avoiding the stigma of first-generation biofuel affecting global food security.
The natural xylose-consumable yeast strain, Candida magnoliae, plays an important role in ethanol and xylitol co-production and could significantly degrade the inhibitors being generated during the pretreatment process when compared with another counterpart strain such as C. tropicalis [8,9]. Bioethanol production under anaerobic conditions could be achieved through a glycolysis pathway by converting glucose to pyruvate by involving a series of enzymes before the subsequent decarboxylation stage to acetaldehyde by pyruvate decarboxylase (PDC, EC 4.1.1.1). Acetaldehyde is then reduced into ethanol by alcohol dehydrogenase (ADH, EC 1.1.1.1) using NADH as a co-substrate [10,11]. Xylitol could be attained by a reduction step of xylose to xylitol via xylose reductase (XR, E.C.1.1.1.21) with either NADH or NADPH as co-factors. Furthermore, xylitol could be subsequently converted to xylulose by the action of xylitol dehydrogenase (XDH, E.C.1.1.1.9) with NAD+ as the co-factor. Phosphorylated xylulose can be utilized by the pentose phosphate pathway for the production of ethanol [12,13].
Cell recycling is an additional strategy for enhancing productivity and product concentration while minimizing capital costs. Generally, recycled cells are utilized in a continuous bioethanol production process with or without cell immobilization. The most favorable separation techniques to obtain recycled cells are membrane filtration and centrifugation. Although the continuous membrane column offers effective separation and high productivity, it is pricey and requires high-energy-consuming steps compared with batch processes [14,15,16]. Five-pass batch ethanol fermentations were proposed with the maximum ethanol concentration of 47.0 g/L (87.4% of the theoretical yield) [17], which could improve productivity up to threefold (~200%) in the fifth batch when compared with the first batch of recycling. An increase in double-recycled cells could improve the ethanol concentration and productivity in the range of 65.5–65.7% in continuous ethanol fermentation [18]. A multiple-pass cell recycling process has been proposed earlier for ethanol and xylitol co-production. Kluyveromyces marxianus was employed with sugarcane bagasse hydrolysate obtained from sulfuric acid pretreatment and enzymatic hydrolysis steps. The application of multiple-pass cell recycling of this microbe revealed the relatively high ethanol productivity of 0.9 ± 0.1 g/L/h with an average ethanol yield of 0.445 ± 0.004 g/g, while xylitol yield was recorded at 0.315 ± 0.01 g/g [19].
This study investigated the application of recycled cells from the wild type of C. magnoliae TISTR 5664 in the single-stage and sequential-stage co-production of xylitol and ethanol with monitoring of mass balance. Corn cob xylose-rich and glucose-rich hydrolysates were used as carbon sources without a detoxification process. Additionally, the degradation of inhibitors generated during the pretreatment process was observed in various xylitol and ethanol production strategies. The novelty of the sequential-stage co-production employing a cell recycling strategy was adapted from sequential aerobic and anaerobic cultivations with cell recycling to substantially enhance the production of xylitol and ethanol. This strategy is designed to circumvent the drawback of conventional two-stage simultaneous saccharification and fermentation (SSF), which generally yields relatively low product concentrations and yields due to limited substrate availability and inefficient cell utilization in a single-pass process.

2. Materials and Methods

2.1. Materials

Corn cob was supported by the Chiang Mai Provincial Livestock Office, Thailand. The pulverized powder (<2 mm) of corn cob was prepared and kept as described by Porninta et al. [8]. Fresh inoculum of C. magnoliae TISTR 5664, a microbial strain obtained from the Thailand Institute of Scientific and Technological Research (TISTR), was propagated as described by Porninta et al. [8]. The commercial enzyme was supplied by Qingdao Vland Biotech Group Co., Ltd., Qingdao, China), with a filter paper activity of 103 ± 0.3 FPU/mL [20]. All chemicals used were analytical grade, excluding calcium hydroxide for neutralization, which was commercial grade.

2.2. Xylose-Rich and Glucose-Rich Hydrolysates Preparation

Corn cob with the highest sugar yield was selected to produce xylose-rich and glucose-rich hydrolysates, as described in our previous publication [8]. Briefly, the pulverized powder of corn cob was pretreated under a diluted sulfuric acid concentration of 3.89% (w/v) for 112 min at 95 ± 1 °C with the liquid-to-solid ratio (LSR) of 10:1 (v/w). The pretreated mixture was then separated using two-layer muslin clothes. The solid fraction was washed with running tap water until the pH reached 4–5 before being dried in a hot air oven (LDO-100E, LabTech, Hwado-eup, Republic of Korea) at 60 °C to the constant weight. The dried solid fraction was hydrolyzed using 45 FPU/g substrate of the commercial enzyme mixtures under the pretreated solid concentration of 12.0% (w/v), 50 °C for 90 h at 200 rpm. After the cessation of the enzyme reaction by vigorous boiling, the hydrolyzed slurry was subsequently separated using the centrifugation technique (modified from Qi et al. [21]). The xylose-rich hydrolysate was obtained from the liquid fraction after acid hydrolysis, while the glucose-rich fraction was the liquid fraction harvested from subsequent enzymatic hydrolysis. These liquids were subsequently evaporated to achieve the optimal concentrations of 50 g/L xylose and 100 g/L glucose, respectively, under 70 °C. Both hydrolysates were adjusted to pH 6 using calcium hydroxide. After pH adjustment, the calcium sulfate precipitate was then removed using centrifugation prior to sterilization at 110 °C for 20 min. The remaining lignin-rich solid fraction after enzymatic hydrolysis was kept in dried form for future utilization.

2.3. Xylitol and Ethanol Co-Production Based on Single-Pass Nonrecycled Cell (Control) Cultivation Process

Xylitol and ethanol co-production was carried out using 10% (v/v) fresh inoculum of C. magnoliae TISTR 5664 with a total working volume of 100 mL in 250 mL nonbaffled Erlenmeyer flasks at 30 °C in an orbital shaker (LSI-3016R, LabTech, Hwado-eup, Republic of Korea). Xylitol was produced using xylose-rich hydrolysate under microaerobic conditions with a rotation speed of 200 rpm in gauze-plugged flasks. Ethanol was produced using glucose-rich hydrolysate under partially anaerobic conditions in screw cap flasks at the rotation speed of 100 rpm. The microbial biomass being produced in this single-pass cell cultivation process will not be reused or recycled, and these processes for producing xylitol and ethanol were treated as controls. The experiments were carried out with quintuplicates, and samples were collected at every 24 h time interval until 240 h.

2.4. Single-Stage Co-Production of Xylitol and Ethanol Using Recycled Cells in One Substrate System

The cells of C. magnoliae TISTR 5664 that were recycled from the single-pass cultivation process were, respectively, inoculated into xylose-rich and glucose-rich hydrolysates for producing xylitol and ethanol. This was called the “First Round”. At a proper cultivation time in the process of the “First Round”, yeast cells were harvested and separated by the refrigerated centrifuge machine (Velocity 18R, Dynamica, Livingston, UK) with a rotation speed of 4170× g for 10 min at 4 °C (modified from Mejias-Ortiz et al. [22]). The harvested cells were used as seed inoculums for the respective xylitol and ethanol productions (“Second Round”) under the same conditions as those described in the control experiment section.

2.5. Sequential-Stage Co-Production of Xylitol and Ethanol Using Recycled Cells in Two Substrates System

Firstly, C. magnoliae TISTR 5664 was inoculated into xylose-rich hydrolysate for xylitol production under microaerobic conditions as mentioned previously. At a proper cultivation time, the yeast cells were harvested and separated using the same refrigerated centrifugation to inoculate into glucose-rich hydrolysate for ethanol production. The proper cultivation time for harvesting cells referred to the time during which maximum xylitol concentration was produced with the shortest cultivation time. The conditions were shifted to partially anaerobic conditions, as mentioned in the control experiment. All treatments were carried out using a similar working volume as the control experiment section.

2.6. Analytical Methods

Sugars (glucose, xylose, and arabinose), xylitol, and ethanol, as well as inhibitor (furfural, 5-hydroxymethylfurfural (5-HMF) and acetic acid) concentrations were analyzed by high-performance liquid chromatography (HPLC) [8]. Dried biomass was assessed using the methodology that Leksawasdi et al. [23] previously reported. Cell viability of yeast was measured using a microscope (CX23, Olympus, Tokyo, Japan) with a hemocytometer [24,25]. The following kinetic parameters, namely yields (Y), maximum specific growth rate (μmax), maximum specific substrate consumption rate (qs,max), maximum product formation rate (qp,max), and maximum productivity (Qmax), were computed using previously described methods [26]. The percentages of theoretical yields (Ythe) of xylitol (YXy/Xyl,the) and ethanol (YEt/TotS,the), which are 0.912 g xylitol/g xylose [27] and 0.511 g ethanol/g total sugars [28], were used for comparison with actual yields. Mass balance calculations of xylitol and ethanol in various strategies based on 1000 g of corn cob were evaluated to assess the efficiency of the process [29]. The density of diluted sulfuric acid (3.89% (w/v)) at 25 °C was obtained from online resources [30].

2.7. Statistical Analysis

The experiments were carried out with quintuplicates and represented as average value ± standard error. The statistically significant difference (p ≤ 0.05) of each experimental set was evaluated using SPSS 22.0 (SPSS, Chicago, IL, USA) run on the Windows Application. Post hoc (Duncan) and ANOVA tests were performed with a similar p-value probability.

3. Results

3.1. Xylitol and Ethanol Co-Production Based on Single-Pass Nonrecycled Cell (Control) Cultivation Process

In the control experiment of the xylitol production process with single-pass cells, xylose-rich hydrolysate was used as the carbon source. The initial glucose, xylose, arabinose, and total sugar concentrations in this hydrolysate were 9.36 ± 0.10, 51.2 ± 1.7, 15.2 ± 0.4, 74.0 ± 1.0 g/L, respectively. C. magnoliae TISTR 5664 from a fresh inoculum rapidly consumed glucose in 24 h. A total of 94.2–94.6% of xylose was utilized between 72 and 120 h, during which xylitol was produced at the maximum extent. Arabinose was gradually decreased after glucose depletion with relatively diminutive xylose remaining, which was consumed by 40.5% and 68.9% in 72 and 120 h, respectively. At the end of the cultivation time (240 h), 95.8% of the total sugars was depleted under the microaerobic conditions. The statistically significant maximum (p ≤ 0.05) xylitol concentration was produced within the range of 28.7 ± 0.4–29.3 ± 1.7 g/L, which corresponded to YXy/Xyl of 0.661 ± 0.008–0.669 ± 0.038 gXy/gXyl (72.5 ± 0.9–73.4 ± 1.2% of YXy/Xyl,the). At the same time interval, ethanol concentration of 2.73 ± 0.26–4.62 ± 0.38 g/L was produced as a byproduct, which corresponded to YEt/TotS of 0.042 ± 0.004–0.078 ± 0.005 gEt/gTotS (8.19 ± 0.73–15.2 ± 0.5% of YEt/TotS,the). Dried biomass was generated at 4.22 ± 0.14–9.19 ± 0.33 g/L, which corresponded to YX/TotS of 0.07 ± 0.01–0.13 ± 0.01 gX/gTotS. However, the statistically significant maximum (p ≤ 0.05) ethanol concentration of 5.59 ± 0.42 g/L, which corresponded to YEt/TotS of 0.120 ± 0.009 gEt/gTotS (23.6 ± 1.8% of YEt/TotS,the), occurred at 48 h. This was compared with the statistically significant maximum (p ≤ 0.05) dried biomass concentration of 17.2 ± 0.3 g/L (0.235 ± 0.006 gX/gTotS of YX/TotS) in 240 h. The kinetic profiles of initial sugars, xylitol, ethanol, and dried biomass concentrations are shown in Figure 1a. In addition, the separated dried biomass concentration kinetic profiles were separately drawn in the Supplementary Materials Section for clarity.
For the ethanol control experiment, glucose-rich hydrolysate with the initial glucose, xylose, and total sugars of 105 ± 1, 27.8 ± 0.3, and 132 ± 1 g/L, respectively, was utilized for this cultivation. Similar to the xylitol control experiment, a fresh inoculum of C. magnoliae TISTR 5664 was employed. The results indicate that under the partially anaerobic condition, 97.5–98.8% of glucose, 53.6–56.8% of xylose, and 88.6–89.9% of total sugars were depleted at the region of statistically significant maximum (p ≤ 0.05) for ethanol production for 120–240 h. In these conditions, only half of the xylose was utilized for the commencement of xylitol and ethanol production. The statistically significant maximum (p ≤ 0.05) range of ethanol concentration was 46.3 ± 1.0–49.3 ± 1.8 g/L, which corresponded to YEt/TotS of 0.450 ± 0.003–0.465 ± 0.010 gEt/gTotS (88.2 ± 2.4–91.1 ± 2.0% of YEt/TotS,the). Xylitol was also produced as a byproduct with a statistically significant maximum (p ≤ 0.05) concentration range of 5.19 ± 0.12–5.51 ± 0.15 g/L, which corresponded to YXy/Xyl of 0.417 ± 0.012–0.456 ± 0.010 gXy/gXyl (45.8 ± 1.4–50.0 ± 1.1% of YXy/Xyl,the) at 144–240 h. Dried biomass was slightly produced at 1.00 ± 0.13–1.30 ± 0.10 g/L (0.008 ± 0.001–0.011 ± 0.001 gX/gTotS of YX/TotS). This was compared with the statistically significant maximum (p ≤ 0.05) dried biomass concentration of 1.41 ± 0.11–1.61 ± 0.05 g/L (0.014 ± 0.001–0.018 ± 0.001 gX/gTotS of YX/TotS) at 48–96 h, as indicated in Figure 2a. The biomass generation was 10-fold lower when compared with the results of the xylitol control experiment.

3.2. Single-Stage Co-Production of Xylitol and Ethanol Using Recycled Cells in One Substrate System

In xylitol production using recycled cells, xylose-rich hydrolysate with the same sugar concentration level as described in the control experiment section was also employed in this section. The recycled cells of C. magnoliae completely consumed glucose in 24 h. Xylose was then rapidly consumed by 80.4–95.0%, while arabinose was consumed at a slower extent by 52.4–71.4% during the maximum xylitol production time span (48–168 h). In fact, 92.2% of total sugars were consumed in 240 h. Evidently, the recycled cells could significantly improve (p ≤ 0.05) ethanol concentration statistically, resulting in the maximum ethanol concentration of 12.6 ± 0.7–13.1 ± 1.0 g/L, which corresponded to YEt/TotS of 0.208 ± 0.014–0.236 ± 0.018 gEt/gTotS (40.7 ± 2.8–46.3 ± 3.5% of YEt/TotS,the) in 48–72 h. This was an improvement of more than twofold compared with ethanol being produced in the xylitol control experiment section. In contrast, the statistically significant maximum (p ≤ 0.05) xylitol concentration was decreased to 10.7 ± 0.8–11.9 ± 0.4 g/L, which corresponded to YXy/Xyl of 0.238 ± 0.018–0.316 ± 0.011 gXy/gXyl (26.1 ± 1.9–34.6 ± 1.2% of YXy/Xyl,the) in 48–168 h. The results indicate that up to 2.5-fold of the xylitol concentration disappeared compared with the xylitol control experiment. At this time interval, ethanol was also generated in the ranges of 5.26 ± 0.29–13.1 ± 1.0 g/L, which corresponded to YEt/TotS of 0.080 ± 0.004–0.236 ± 0.018 gEt/gTotS (15.6 ± 0.8–46.3 ± 3.5% of YEt/TotS,the). At this time interval, ethanol was also generated within the ranges of 7.79 ± 0.29–11.0 ± 0.25 g/L (0.118 ± 0.006–0.159 ± 0.002 gX/gTotS of YX/TotS). The statistically significant maximum (p ≤ 0.05) dried biomass concentration was 14.3 ± 0.1 g/L (0.205 ± 0.011 gX/gTotS). The kinetic profiles of initial sugars, xylitol, ethanol, and dried biomass concentrations using xylose-rich hydrolysate and recycled cells are shown in Figure 1b.
Similar to the xylitol production using the recycled yeast cells, employing the recycling cells with glucose-rich hydrolysate in ethanol production demonstrated that the maximum ethanol concentration was significantly (p ≤ 0.05) higher than the ethanol control experiment by 7.8%. Figure 2b shows all kinetic profiles of this cultivation: 86.2%–96.0% of glucose and 38.8%–46% of xylose were consumed during the maximum ethanol production time span (144–240 h). A total of 85.8 ± 1.1% of the total sugars was used up at the end of cultivation time. The statistically significant maximum (p ≤ 0.05) ethanol concentration was in the range of 49.9 ± 1.3–51.7 ± 1.7 g/L, which corresponded to YEt/TotS of 0.472 ± 0.006–0.487 ± 0.009 gEt/gTotS (92.5 ± 1.2–95.6 ± 1.7% of YEt/TotS,the) with no xylitol production. A dried biomass concentration of 1.48 ± 0.06–1.75 ± 0.07 g/L (0.014 ± 0.001–0.017 ± 0.002 gX/gTotS of YX/TotS) was generated at the same time. In fact, the statistically significant maximum (p ≤ 0.05) of dried biomass accumulated to 2.16 ± 0.11 g/L (0.040 ± 0.003 gX/gTotS of YX/TotS) at 48 h.

3.3. Sequential-Stage Co-Production of Xylitol and Ethanol Using Recycled Cells in Two Substrates System

The first step production was carried out for 72 h, which was the shortest optimal xylitol production time in the xylitol control experiment. Xylose-rich hydrolysate and fresh inoculum of C. magnoliae were employed for xylitol production in this stage. The statistically significant maximum (p ≤ 0.05) xylitol concentration at 72 h was achieved at 25.9 ± 0.5 g/L, which corresponded to YXy/Xyl of 0.534 ± 0.012 gXy/gXyl (58.6 ± 1.3% of YXy/Xyl,the). Ethanol concentration in this stage was in the range of 12.4 ± 0.7–13.2 ± <0.1 g/L, which corresponded to YEt/TotS of 0.194 ± 0.005–0.198 ± 0.018 gEt/gTotS (38.0 ± 0.9–38.8 ± 1.6% of YEt/TotS,the). The dried biomass concentration was 6.10 ± 0.19 g/L (0.088 ± 0.003 gX/gTotS). After 72 h, the second-step production was established using the recycled yeast cells harvested from the first-step production. The conditions were changed to ethanol production as described in the Materials and Methods Section. The statistically significant maximum (p ≤ 0.05) ethanol concentration was in the range of 50.9 ± 1.0–54.1 ± 2.1 g/L, which corresponded to YEt/TotS of 0.394 ± 0.007–0.420 ± 0.014 gEt/gTotS (77.3 ± 1.4–83.9 ± 3.0% of YXy/Xyl,the) in 264–312 h. In this stage, a lack of xylitol production was observed, while dried biomass concentration was slightly decreased to 4.67 ± 0.16–4.94 ± 0.14 g/L (0.034 ± 0.002–0.037 ± 0.002 gX/gTotS). The achievable ethanol concentrations after the end of the first and second steps were 12.4 ± 0.7–13.2 ± <0.1 and 50.9 ± 1.0–54.1 ± 2.1 g/L, as shown in Figure 3, which indicated an approximately 310% improvement on the second step of ethanol production in relation to the first step of xylitol production.

3.4. Comparative Co-Production of Xylitol and Ethanol in Single-Stage and Sequential-Stage

Employing a cell recycling strategy resulted in ethanol production improvement. In such a system, ethanol concentration was raised from the original value of 7.8% (49.9 ± 1.3–51.7 ± 1.7 g/L) in the single-stage to 9.9% (50.9 ± 1.0–54.1 ± 2.1 g/L) when the sequential-stage was employed. The accumulation of NAD+ in the recycled yeast cells grown under partially anaerobic conditions [31] might be the cause of proliferation in ethanol concentration. Xylose reductase enzyme can reduce xylose to xylitol, which will be converted in turn to xylulose by xylitol dehydrogenase with NAD+ as a co-enzyme. This compound is then used as a substrate for ethanol production [12,13]. Evidently, there is no xylitol being detected in all ethanol production strategies, as it may be immediately consumed in succession from xylitol to ethanol. The most practical method in the developed sequential-stage co-production of xylitol and ethanol was to produce xylitol initially so that sufficient cells and NAD+ accumulation could be achieved for the subsequent steps of ethanol production. In xylitol production under microaerobic conditions, C. magnoliae TISTR 5664—the natural xylose utilizer—could produce the highest (p ≤ 0.05) xylitol concentration. Moreover, under the aerated conditions, accumulated yeast cells were observed due to the utilization of oxygen and sugars to generate ATP in the process of oxidative phosphorylation [11]. The harvested yeast cells with the accumulated NAD+ help facilitate ethanol production without xylitol as a byproduct in the second step. Seed propagation and cultivation preparation are not required in this second step due to the readily available recycled cells.
The monitoring of viable cells was also carried out during xylitol and ethanol production in all present studies (Figure 4). The relatively high cell viability persisted in the range of 91.2–99.5% under aerated conditions for 240 h in the xylitol production stage. Progressive cell death was observed at 192 h, both in single-stage ethanol control and recycling cell experiments under partially anaerobic conditions, with cell viabilities of 14.2 ± 0.5% and 20.9 ± 1.9%, respectively, for 48 h until 240 h. In sequential-stage co-production, dead cells tended to increase from 264 h to 312 h (48 h interval), with 24.4 ± 2.1% cell viability. Table 1 indicates xylitol, ethanol, and dried biomass productions with the corresponding kinetic parameters, including cell viability.

3.5. Investigation of the Inhibitors’ Degradation

C. magnoliae TISTR 5664 could significantly mitigate the inhibitor concentrations being formed during the acid pretreatment process in a xylose-rich hydrolysate. A decrease in 5-HMF by 96.8% (a drop from 394 ± 5 to 12.8 ± 0.9 mg/L) was detected in the xylitol control experiment. This was compared with the case of the xylitol production system utilizing recycled cells, where a deduction of 98.0% (394 ± 5 to 7.95 ± 0.30 mg/L) 5-HMF was observed after 24 h (Figure 5(a1)). For furfural degradation kinetics, 77.2% (47.3 ± 1.3 to 10.8 ± 0.3 mg/L) was decreased in the xylitol control experiment and 65.5% (47.3 ± 0.5 to 16.3 ± 0.4 mg/L) was diminished in the recycled cell counterpart (Figure 5(b1)) at 24 h. In the case of ethanol production using glucose-rich hydrolysate after the enzymatic hydrolysis process, both 5-HMF and furfural could not be detected. Additionally, C. magnoliae could totally degrade acetic acid in the xylitol control experiment, while recycled cells could deduct 93.0% (4.20 ± 0.03 to 0.31 ± 0.03 g/L) of this acid at 240 h, as indicated in Figure 5(c1). In fact, acetic acid concentration in the ethanol control experiment elevated by 45.7% (from 6.48 ± 0.11 to 9.44 ± 0.60 g/L) after employing the cell recycling strategy. It should be noted that the acetic acid concentration profile was relatively constant during the ethanol control experiment (Figure 5(c1)). Such an occurrence could be the result of the acetate-to-ethanol reverse conversion pathway. Some ethanol could be oxidized to acetaldehyde and subsequently transformed to acetic acid as a byproduct [10,32]. Evidently, the acetic acid being formed in the single-stage production during the ethanol route with recycled cells was raised to 9.44 ± 0.60 g/L after 240 h cultivation time. Furthermore, in the sequential-stage cultivation systems, the concentration of acetic acid in the ethanol production step increased by more than 32.7% (12.5 ± 0.1 g/L) compared with the single-stage system mentioned previously.

3.6. Mass Balance Calculations of Xylitol and Ethanol in Various Production Strategies

The 1 kg basis of corn cob mass balance calculations on xylitol and ethanol production are presented in Figure 6. The initial composition of principal components, namely cellulose, hemicellulose, and lignin in corn cob was taken directly from Porninta et al. [8]. After diluted sulfuric acid pretreatment, about 50–56% (w/w) of the pretreated solid was obtained, along with the liquid fraction (xylose-rich hydrolysate) to be used for the xylitol production stage. The hemicellulose content in the pretreated solid was dropped from 38.3 ± 0.4 to 5.95 ± 0.18% (w/w) (29.8 ± 0.9 g in Figure 6), as it was digested into the liquid fraction with 37.5 ± 1.0 g glucose, 207 ± <1 g xylose, and 74.8 ± 1.2 g arabinose. The pretreated solid fraction was further hydrolyzed by a commercial cellulase/xylanase enzyme mixture. After centrifugation, the liquid fraction (glucose-rich hydrolysate) contained 216 ± 2 g glucose and 44.5 ± 0.4 g xylose. The statistically significant maximum (p ≤ 0.05) xylitol production was achieved at 115 ± 2–117 ± 3 g from the xylitol control experiment. In fact, the statistically significant maximum (p ≤ 0.05) ethanol of 105 ± 3–109 ± 3 g was obtained from single-stage and sequential-stage co-production using recycled cells. In the first step (0–72 h) of developed sequential-stage co-production, xylitol and ethanol could be produced at 104 ± 2 and 49.6 ± 2.8–52.8 ± <0.1 g, respectively. The statistically significant maximum (p ≤ 0.05) level of ethanol at 107 ± 2–114 ± 4 g could be achieved during the second-step production (72–312 h). The overall ethanol from sequential-stage production was estimated at 157 ± 3–167 ± 4 g.

4. Discussion

The developed sequential-stage co-production resulted in higher xylitol and ethanol concentrations when compared with Cheng et al. [9]. That study described the prospective of the sequential aerobic and anaerobic processes for xylitol and ethanol production with the anaerobic SSF using C. tropicalis W103. Under the optimum substrate loading, the maximum xylitol and ethanol concentrations were reported at 17.1 g/L and 25.3 g/L, which corresponded to 35.1% and 82.5% of the theoretical yield, respectively. A limited substrate loading was the cause of the rather low xylitol and ethanol concentrations, as stated by Cheng et al. [9]. The sequential-stage co-production process employing recycled cells in this study was developed to solve this challenge by increasing the concentration of initial sugars to obtain statistically significant higher (p ≤ 0.05) xylitol and ethanol concentrations [33,34,35]. Furthermore, the potential of multiple-pass cell recycling was illustrated by Dasgupta et al. [19]. In that report, the sole production of xylitol and ethanol could be achieved by multiple-pass cell recycling of K. marxianus IIPE453 with relatively low concentrations of 11.1 ± 0.07 g/L xylitol and 21.6 g/L ethanol. These corresponded to 0.315 ± 0.01 g/g of YXy/Xyl and 0.445 ± 0.004 g/g of YEt/Glu, respectively, for the average of triple-pass cell recycling.
The results of inhibitor degradation in the external control experiment were comparable to our previously published report [8] in a similar system. Cheng et al. [9], likewise, found that C. tropicalis W103 expressed a tolerant endurance to completely degrade 5-HMF and furfural within 60 h, while 89.4% of the acetic acid was utilized during the xylitol production process. For sequential-stage co-production kinetics, 5-HMF, furfural, and acetic acid degradation profiles are displayed in Figure 5(a2), Figure 5(b2), and Figure 5(c2), respectively. The similar kinetics trends of 5-HMF and furfural could be observed from both control experiments and cell recycling processes. Thus, the detoxification process was not crucial for a cost-effective strategy in comparison with the employment of single-stage control experiments and recycled cells, as proposed in this study and previously published papers [8,9,36,37].
The comparison of yields from a multiple passes cells recycling process [19] revealed the relatively lower values of 62.5 g xylitol and 145 g ethanol on a 1 kg basis of sugarcane bagasse. Furthermore, this study could improve 42% and 199–218% of xylitol and ethanol based on the similar dry basis of pretreated corn cob in a fed-batch two-stage fermentation strategy [36].

5. Conclusions

The novel adaptation of the cell recycling strategy in the xylitol and ethanol sequential-stage co-production system by the efficient usage of cultivated C. magnoliae TISTR 5664 could produce both products with relatively reasonable concentrations and yields based on a single round of cell inoculation compared with previous reports of a similar system. Such a process could maximize productivity by eliminating the time required for seed propagation and cultivation preparation. The benefits of applying a cell recycling strategy to both single-stage and sequential-stage production of ethanol were also strongly pronounced with equivalent optimal concentrations and yields. These results may enhance the possibility of integrating agricultural waste with the synthesis of other high-value compounds to establish a more sustainable bioeconomy from a socioecological perspective. In order to evaluate its efficacy for industrial-scale production and address growing environmental challenges like climate change and the detrimental impact of air pollution on agricultural productivity, additional inquiry on a larger-scale system for sequential-stage co-production with a benefit–cost ratio should be clarified later.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture14071062/s1, Figure S1: Kinetic profiles of dried biomass concentrations during xylitol production under partially anaerobic conditions; (a) nonrecycled cell (control) cultivation process; (b) single-stage production using recycled cells.; Figure S2: Kinetic profiles of dried biomass concentrations during ethanol production under partially anaerobic conditions; (a) nonrecycled cell (control) cultivation process; (b) single-stage production using recycled cells.; Figure S3: Kinetic profiles of dried biomass concentrations during sequential-stage co-production; the first step: xylitol production using fresh inoculum under microaerobic conditions; the second step: ethanol production using recycled cells obtained from the first step under partially anaerobic conditions.

Author Contributions

Conceptualization, K.P., R.N., S.S. and N.L.; methodology, K.P., C.M. and S.S.; validation, K.P., C.M., S.S. and N.L.; formal analysis, K.P., J.K., W.W., W.Q. and S.S.; investigation, K.P., C.M., J.F., S.L.H., S.S. and N.L.; resources, S.S. and N.L.; data curation, K.P., S.S. and N.L.; writing—original draft preparation, K.P.; writing—review and editing, K.P., J.K., C.T., Y.P., P.R., K.J., J.F., S.L.H., R.N., X.Z., W.W., W.Q., Z.W., S.S. and N.L.; supervision, Z.W.; project administration, N.L.; funding acquisition, K.P., C.T., Y.P., P.R., K.J., S.S. and N.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Research Council of Thailand (NRCT) in the framework of the Royal Golden Jubilee (RGJ) Ph.D. Programme (Grant Number: PHD/0111/2560); Fundamental Fund 2024 by Thailand Science Research and Innovation (TSRI) (Grant Number: FF037/2567); Chiang Mai University (CMU) Proactive Postmaster Researcher (Grant Number: 881/2566); Center of Excellence-Agro Bio-Circular-Green Industry (Agro-BCG) (Grant Number: CoE66-P001); Thailand Research Fund (TRF) Research Team Promotion Grant, RTA, Senior Research Scholar (N42A671052).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data sets generated and/or analyzed during the current study are available from the corresponding authors upon reasonable request.

Acknowledgments

This work was supported by the National Research Council of Thailand (NRCT) in the framework of the Royal Golden Jubilee (RGJ) Ph.D. Programme to K. Porninta and N. Leksawasdi; Fundamental Fund 2024 provided by Thailand Science Research and Innovation (TSRI); Chiang Mai University (CMU) Proactive Postmaster Researcher; Center of Excellence-Agro Bio-Circular-Green Industry (Agro-BCG); Multidisciplinary and Interdisciplinary School, CMU; School of Agro-Industry and Faculty of Agro-Industry, CMU; Bioprocess Research Cluster (BRC); Office of Research Administration (ORA). The present study was partially supported by the Thailand Research Fund (TRF) Research Team Promotion Grant, RTA, Senior Research Scholar. Funding numbers are provided in the Funding Section. TISTR is also thanked for microbial strain support.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations and Symbols

[ ]Concentration
μmaxMaximum specific growth rate (h−1)
EtEthanol
FPUFilter paper units
GluGlucose
5-HMF5-Hydroxymethylfurfural
n.d.Not detected
QEt,maxVolumetric productivity of ethanol per liter per h (gEt/L/h)
QXy,maxVolumetric productivity of xylitol per liter per h (gXy/L/h)
qEt,maxMaximum specific ethanol production rate (gEt/gX/h)
qTotS,maxMaximum specific total sugars consumption rate (gTotS/gX/h)
qXy,maxMaximum specific xylitol production rate (gXy/gX/h)
TISTRThailand Institute of Scientific and Technological Research
TotSTotal sugars
XDried biomass
XyXylitol
XylXylose
YEt/TotSYield of ethanol produced over total sugars consumed (gEt/gTotS)
YX/TotSYield of dried biomass produced over total sugars consumed (gX/gTotS)
YXy/XylYield of xylitol produced over xylose consumed (gXy/gXyl)
YtheTheoretical yields.

References

  1. International Energy Agency (IEA). Share of Global Ethanol Output by Country between 2017 and 2023. Available online: https://www.iea.org/data-and-statistics/charts/share-of-global-ethanol-output-by-country-between-2017-and-2023 (accessed on 20 March 2024).
  2. United States Department of Energy. Global Ethanol Production by Country or Region. Available online: https://afdc.energy.gov/data/10331 (accessed on 17 March 2024).
  3. Dahman, Y.; Dignan, C.; Fiayaz, A.; Chaudhry, A. An introduction to biofuels, foods, livestock, and the environment. In Biopolymer-Based Materials, and Bioenergy; Woodhead Publishing Series in Composites Science and Engineering; Woodhead Publishing: Cambridge, UK, 2019; pp. 241–276. [Google Scholar] [CrossRef]
  4. Shokravi, H.; Shokravi, Z.; Heidarrezaei, M.; Ong, H.C.; Koloor, S.S.R.; Petrů, M.; Lau, W.J.; Ismail, A.F. Fourth generation biofuel from genetically modified algal biomass: Challenges and future directions. Chemosphere 2021, 285, 131535. [Google Scholar] [CrossRef]
  5. Ale, S.; Femeena, P.V.; Mehan, S.; Cibin, R. Environmental impacts of bioenergy crop production and benefits of multifunctional bioenergy systems. In Bioenergy with Carbon Capture and Storage; Academic Press: New York, NY, USA, 2019; pp. 195–217. [Google Scholar] [CrossRef]
  6. Mathur, S.; Kumar, D.; Kumar, V.; Dantas, A.; Verma, R.; Kuca, K. Xylitol: Production strategies with emphasis on biotechnological approach, scale up, and market trends. Sustain. Chem. Pharm. 2023, 35, 101203. [Google Scholar] [CrossRef]
  7. ReportLinker. Prospective Insights into Xylitol Sector: 2023 Global Review. Available online: https://www.reportlinker.com/p06284602/Xylitol-Global-Market-Report.html?utm_source=GNW (accessed on 20 March 2024).
  8. Porninta, K.; Khemacheewakul, J.; Techapun, C.; Phimolsiripol, Y.; Jantanasakulwong, K.; Sommanee, S.; Mahakuntha, C.; Feng, J.; Htike, S.L.; Kumar, A.; et al. Pretreatment and Enzymatic Hydrolysis Optimization of Lignocellulosic Biomass for Ethanol, Xylitol, and Phenylacetylcarbinol Co-Production using Candida magnoliae. Front. Bioeng. Biotechnol. 2024, 11, 2023. [Google Scholar] [CrossRef]
  9. Cheng, K.; Wu, J.; Lin, Z.; Zhang, J. Aerobic and sequential anaerobic fermentation to produce xylitol and ethanol using non-detoxified acid pretreated corncob. Biotechnol. Biofuels 2014, 7, 166. [Google Scholar] [CrossRef]
  10. Eknikom, S.; Nasuno, R.; Takagi, H. Molecular mechanism of ethanol fermentation inhibition via protein tyrosine nitration of pyruvate decarboxylase by reactive nitrogen species in yeast. Sci. Rep. 2022, 12, 4664. [Google Scholar] [CrossRef]
  11. Pfeiffer, T.; Morley, A. An evolutionary perspective on the Crabtree effect. Front. Mol. Biosci. 2014, 1, 17. [Google Scholar] [CrossRef]
  12. Baptista, S.L.; Cunha, J.T.; Romaní, A.; Domingues, L. Xylitol production from lignocellulosic whole slurry corn cob by engineered industrial Saccharomyces cerevisiae PE-2. Bioresour. Technol. 2018, 267, 481–491. [Google Scholar] [CrossRef]
  13. Mareczky, Z.; Anikó, F.; Anikó, F.; Fehér, C.; Réczey, K. Effects of pH and aeration conditionson xylitol production by Candida and Hansenula yeasts. Period. Polytech. Chem. Eng. 2016, 60, 54–59. [Google Scholar] [CrossRef]
  14. Nimbalkar, P.R.; Khedkar, M.A.; Chavan, P.V.; Bankar, S.B. Biobutanol from agricultural residues: Technology and economics. In Advances and Developments in Biobutanol Production; Segovia-Hernandez, J.G., Behera, S., Sanchez-Ramirez, E., Eds.; Woodhead Publishing: Cambridge, UK, 2023; Volume Advances in Pollution Research, pp. 139–169. [Google Scholar]
  15. Trilokesh, C.; Uppuluri, K.B. Biobutanol from lignocellulosic biomass and microalgae: Scope, technology, and economics. In Sustainable Biofuels; Ray, R.C., Ed.; Academic Press: New York, NY, USA, 2021; Volume Applied Biotechnology Reviews, pp. 163–223. [Google Scholar]
  16. Xue, C.; Cheng, C. Butanol production by Clostridium. In Advances in Bioenergy; Li, Y., Ge, X., Eds.; Elsevier: Amsterdam, The Netherlands, 2019; Volume 4, pp. 35–77. [Google Scholar]
  17. Silva, V.F.; Nakanishi, S.C.; Dionísio, S.R.; Rossell, C.E.; Ienczak, J.L.; Gonçalves, A.R.; Rocha, G.J. Using cell recycling batch fermentations to validate a setup for cellulosic ethanol production. J. Chem. Technol. Biotechnol. 2016, 91, 1853–1859. [Google Scholar] [CrossRef]
  18. Thani, A.; Laopaiboon, P.; Laopaiboon, L. Batch and continuous ethanol fermentation by Saccharomyces cerevisiae NP 01 using cell recycling technique. Asia-Pac. J. Sci. Technol. 2017, 19, 1–8. [Google Scholar]
  19. Dasgupta, D.; Ghosh, D.; Bandhu, S.; Adhikari, D.K. Lignocellulosic sugar management for xylitol and ethanol fermentation with multiple cell recycling by Kluyveromyces marxianus IIPE453. Microbiol. Res. 2017, 200, 64–72. [Google Scholar] [CrossRef]
  20. Nunta, R.; Techapun, C.; Sommanee, S.; Mahakuntha, C.; Porninta, K.; Punyodom, W.; Phimolsiripol, Y.; Rachtanapun, P.; Wang, W.; Zhuang, X.; et al. Valorization of rice straw, sugarcane bagasse and sweet sorghum bagasse for the production of bioethanol and phenylacetylcarbinol. Sci. Rep. 2023, 13, 727. [Google Scholar] [CrossRef]
  21. Qi, B.; Chen, X.; Shen, F.; Su, Y.; Wan, Y. Optimization of enzymatic hydrolysis of wheat straw pretreated by alkaline peroxide using response surface methodology. Ind. Eng. Chem. Res. 2009, 48, 7346–7353. [Google Scholar] [CrossRef]
  22. Mejias-Ortiz, M.; Mencher, A.; Morales, P.; Tronchoni, J.; Gonzalez, R. Saccharomyces cerevisiae responds similarly to co-culture or to a fraction enriched in Metschnikowia pulcherrima extracellular vesicles. Microb. Biotechnol. 2023, 16, 1027–1040. [Google Scholar] [CrossRef]
  23. Leksawasdi, N.; Chow, Y.Y.S.; Breuer, M.; Hauer, B.; Rosche, B.; Rogers, P.L. Kinetic analysis and modelling of enzymatic (R)-phenylacetylcarbinol batch biotransformation process. J. Biotechnol. 2004, 111, 179–189. [Google Scholar] [CrossRef]
  24. Arshad, M.; Khan, Z.M.; Khalil-ur-Rehman; Shah, F.A.; Rajoka, M.I. Optimization of process variables for minimization of byproduct formation during fermentation of blackstrap molasses to ethanol at industrial scale. Lett. Appl. Microbiol. 2008, 47, 410–414. [Google Scholar] [CrossRef]
  25. Tangtua, J.; Techapun, C.; Pratanaphon, R.; Kuntiya, A.; Chaiyaso, T.; Hanmoungjai, P.; Seesuriyachan, P.; Leksawasdi, N. Screening of 50 microbial strains for production of ethanol and (R)-phenylacetylcarbinol. Chiang Mai J. Sci. 2013, 40, 299–304. [Google Scholar]
  26. Mahakuntha, C.; Reungsang, A.; Nunta, R.; Leksawasdi, N. Kinetics of whole cells and ethanol production from Candida tropicalis TISTR 5306 cultivation in batch and fed-batch modes using assorted grade fresh longan juice. An. Acad. Bras. Cienc. 2021, 93, e20200220. [Google Scholar] [CrossRef] [PubMed]
  27. Tamburini, E.; Costa, S.; Marchetti, M.G.; Pedrini, P. Optimized production of xylitol from xylose using a hyper-acidophilic Candida tropicalis. Biomolecules 2013, 5, 1979–1989. [Google Scholar] [CrossRef] [PubMed]
  28. Li, Y.; Zhai, R.; Jiang, X.; Chen, X.; Yuan, X.; Liu, Z.; Jin, M. Boosting ethanol productivity of Zymomonas mobilis 8b in enzymatic hydrolysate of dilute acid and ammonia pretreated corn stover through medium optimization, high cell density fermentation and cell recycling. Front. Microbiol. 2019, 10, 2316. [Google Scholar] [CrossRef]
  29. Lee, J.; Kim, S.; Lee, K.H.; Lee, S.K.; Chun, Y.; Kim, S.W.; Park, C.; Yoo, H.Y. Improvement of bioethanol production from waste chestnut shells via evaluation of mass balance-based pretreatment and glucose recovery process. Environ. Technol. Innov. 2022, 28, 102955. [Google Scholar] [CrossRef]
  30. Sunga, R. The Complete Aqueous Sulfuric Acid Solutions Density-Concentration Calculator. Available online: https://www.handymath.com/cgi-bin/sulfurictble11.cgi?submit=Back+to+Calculator&tmptr=25&spcfgrv=&conc=3.89 (accessed on 13 May 2024).
  31. Croft, T.; Venkatakrishnan, P.; Lin, S.-J. NAD+ Metabolism and Regulation: Lessons from Yeast. Biomolecules 2020, 10, 330. [Google Scholar] [CrossRef]
  32. Wei, Y.; Lin, M.; Oliver, D.J.; Schnable, P.S. The roles of aldehyde dehydrogenases (ALDHs) in the PDH bypass of Arabidopsis. BMC Biochem. 2009, 10, 7. [Google Scholar] [CrossRef] [PubMed]
  33. Chang, Y.-H.; Chang, K.-S.; Chen, C.-Y.; Hsu, C.-L.; Chang, T.-C.; Jang, H.-D. Enhancement of the efficiency of bioethanol production by Saccharomyces cerevisiae via gradually batch-wise and fed-batch increasing the glucose concentration. Fermentation 2018, 4, 45. [Google Scholar] [CrossRef]
  34. Narisetty, V.; Castro, E.; Durgapal, S.; Coulon, F.; Jacob, S.; Kumar, D.; Awasthi, M.K.; Pant, K.K.; Parameswaran, B.; Kumar, V. High level xylitol production by Pichia fermentans using non-detoxified xylose-rich sugarcane bagasse and olive pits hydrolysates. Bioresour. Technol. 2021, 342, 126005. [Google Scholar] [CrossRef] [PubMed]
  35. Qazizada, M.E. Design of a batch stirred fermenter for ethanol production. Procedia Eng. 2016, 149, 389–403. [Google Scholar] [CrossRef]
  36. Du, C.; Li, Y.; Zong, H.; Yuan, T.; Yuan, W.; Jiang, Y. Production of bioethanol and xylitol from non-detoxified corn cob via a two-stage fermentation strategy. Bioresour. Technol. 2020, 310, 123427. [Google Scholar] [CrossRef]
  37. Ji, H.; Xu, K.; Dong, X.; Sun, D.; Jin, L. Sequential Production of d-xylonate and Ethanol from Non-Detoxified Corncob at Low-pH by Pichia kudriavzevii via a Two-Stage Fermentation Strategy. J. Fungi 2021, 7, 1038. [Google Scholar] [CrossRef]
Agriculture 14 01062 g001
Figure 1. Kinetic profiles of sugars (glucose, xylose, arabinose, and total sugars), xylitol, ethanol, and dried biomass concentrations during xylitol production under microaerobic conditions; (a) nonrecycled cell (control) cultivation process; (b) single-stage production using recycled cells.
Figure 1. Kinetic profiles of sugars (glucose, xylose, arabinose, and total sugars), xylitol, ethanol, and dried biomass concentrations during xylitol production under microaerobic conditions; (a) nonrecycled cell (control) cultivation process; (b) single-stage production using recycled cells.
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Figure 2. Kinetic profiles of sugars (glucose, xylose, and total sugars), xylitol, and ethanol, as well as dried biomass concentrations during ethanol production under partially anaerobic conditions; (a) nonrecycled cell (control) cultivation process; (b) single-stage production using recycled cells.
Figure 2. Kinetic profiles of sugars (glucose, xylose, and total sugars), xylitol, and ethanol, as well as dried biomass concentrations during ethanol production under partially anaerobic conditions; (a) nonrecycled cell (control) cultivation process; (b) single-stage production using recycled cells.
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Figure 3. Kinetic profiles of sugars (glucose, xylose, arabinose, and total sugars), xylitol, ethanol, and dried biomass concentrations during sequential-stage co-production; the first step: xylitol production using fresh inoculum under microaerobic conditions; the second step: ethanol production using recycled cells obtained from the first step under partially anaerobic conditions.
Figure 3. Kinetic profiles of sugars (glucose, xylose, arabinose, and total sugars), xylitol, ethanol, and dried biomass concentrations during sequential-stage co-production; the first step: xylitol production using fresh inoculum under microaerobic conditions; the second step: ethanol production using recycled cells obtained from the first step under partially anaerobic conditions.
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Figure 4. Cell viability of C. magnoliae TISTR 5664 during xylitol and ethanol co-production based on nonrecycled cell (control) cultivation process and single-stage using recycled cells, as well as sequential-stage co-production.
Figure 4. Cell viability of C. magnoliae TISTR 5664 during xylitol and ethanol co-production based on nonrecycled cell (control) cultivation process and single-stage using recycled cells, as well as sequential-stage co-production.
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Figure 5. Kinetics profiles of inhibitors degradation; (a) 5-HMF, (b) furfural, and (c) acetic acid during (a1,b1,c1) xylitol and ethanol co-production based on nonrecycled cell (control) cultivation process and single-stage co-production using recycled cells, as well as (a2,b2,c2) sequential-stage co-production.
Figure 5. Kinetics profiles of inhibitors degradation; (a) 5-HMF, (b) furfural, and (c) acetic acid during (a1,b1,c1) xylitol and ethanol co-production based on nonrecycled cell (control) cultivation process and single-stage co-production using recycled cells, as well as (a2,b2,c2) sequential-stage co-production.
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Figure 6. Mass balance of xylitol and ethanol production for various strategies based on 1000 g of corn cob.
Figure 6. Mass balance of xylitol and ethanol production for various strategies based on 1000 g of corn cob.
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Table 1. Xylitol, ethanol, and dried biomass productions with the corresponding kinetic parameters, including cell viability using xylose-rich and glucose-rich hydrolysates from corn cob without detoxification process as carbon sources.
Table 1. Xylitol, ethanol, and dried biomass productions with the corresponding kinetic parameters, including cell viability using xylose-rich and glucose-rich hydrolysates from corn cob without detoxification process as carbon sources.
[Product],
Kinetic Parameter,
Cells Viability		Xylitol Production Stage
in Xylose-Rich Hydrolysate		Ethanol Production Stage
in Glucose-Rich Hydrolysate		Sequential-Stage Co-Production
ControlSingle-Stage
(Recycled Cells)		ControlSingle-Stage
(Recycled Cells)		1st Step: Xylose-Rich Hydrolysate2nd Step: Glucose-Rich Hydrolysate
(Recycled Cells)
[Xy]max (g/L)28.7–29.3 A10.7–11.9 C5.19–5.51 Dn.d.25.9 Bn.d.
[Et]max (g/L)5.59 D12.6–13.1 C46.3–49.3 B 49.9–51.7 A12.4–13.2 C50.9–54.1 A
[X]max (g/L)17.2 A14.3 B1.41–1.61 E2.16 E6.10 C4.67–4.94 D
YXy/Xyl (gXy/gXyl)0.661–0.669 A0.238–0.316 D0.417–0.456 Cn.d.0.534 Bn.d.
YEt/TotS (gEt/gTotS)0.120 E0.208–0.236 D0.450–0.465 B0.472–0.487 A0.194–0.198 D0.394–0.420 C
YX/TotS (gX/gTotS)0.235 A0.205 B0.014–0.018 E0.040 D0.088 C0.036–0.037 DE
μmax (h−1)0.027 B0.012 C0.028 B0.011 C0.033 A0.003 D
qTotS,max (gTotS/gX/h)−0.86 C−0.18 D−1.63 B−0.66 C−2.18 A−0.30 D
qXy,max (gXy/gX/h)0.59 A0.10 D0.19 C–0.26 B–
qEt,max (gEt/gX/h)0.37 C0.07 D0.85 A0.57 B0.84 A0.10 D
QXy,max (gXy/L/h)0.40–0.41 A0.22–0.25 C0.036–0.038 D–0.36 B–
QEt,max (gEt/L/h)0.12 D0.26–0.27 C0.38–0.41 A0.35–0.36 B0.26–0.27 C0.27–0.28 C
Cell viability * (%)99.3 A91.2 B14.2 D20.9 C99.1 A19.6 C
Xy = xylitol, Et = ethanol, X = dried biomass, Xyl = xylose, TotS = total sugars, n.d. = not detected; [ ], yield (Y), and cell viability in this table are average values from quintuplicates and SE values are less than 10%, while other kinetic parameters were estimated tangentially from the profiles in triplicate; numbers with the same superscript capital alphabet indicate no significant difference (p > 0.05) for comparison of the same row; bolded and underlined values indicated the statistical significantly highest (p ≤ 0.05) in the same row. * cell viability was reported at 240 h, excepting in the first and second steps of the sequential-stage co-production, which were reported at 72 and 312 h, respectively.
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Porninta, K.; Mahakuntha, C.; Khemacheewakul, J.; Techapun, C.; Phimolsiripol, Y.; Rachtanapun, P.; Jantanasakulwong, K.; Feng, J.; Htike, S.L.; Nunta, R.; et al. Cell Recycling Application in Single-Stage and Sequential-Stage Co-Production of Xylitol and Ethanol Using Corn Cob Hydrolysates. Agriculture 2024, 14, 1062. https://doi.org/10.3390/agriculture14071062

AMA Style

Porninta K, Mahakuntha C, Khemacheewakul J, Techapun C, Phimolsiripol Y, Rachtanapun P, Jantanasakulwong K, Feng J, Htike SL, Nunta R, et al. Cell Recycling Application in Single-Stage and Sequential-Stage Co-Production of Xylitol and Ethanol Using Corn Cob Hydrolysates. Agriculture. 2024; 14(7):1062. https://doi.org/10.3390/agriculture14071062

Chicago/Turabian Style

Porninta, Kritsadaporn, Chatchadaporn Mahakuntha, Julaluk Khemacheewakul, Charin Techapun, Yuthana Phimolsiripol, Pornchai Rachtanapun, Kittisak Jantanasakulwong, Juan Feng, Su Lwin Htike, Rojarej Nunta, and et al. 2024. "Cell Recycling Application in Single-Stage and Sequential-Stage Co-Production of Xylitol and Ethanol Using Corn Cob Hydrolysates" Agriculture 14, no. 7: 1062. https://doi.org/10.3390/agriculture14071062

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