Next Article in Journal
A Goal-Oriented Evaluation Methodology for Privacy-Preserving Process Mining
Previous Article in Journal
Explainable Liver Segmentation and Volume Assessment Using Parallel Cropping
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Applied Sciences
Volume 15
Issue 14
10.3390/app15147808
applsci-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Harnessing Carrot Discard as a Novel Feedstock for 2,3-Butanediol Bioproduction: A Comparison of Fermentation Strategies and Bacillus Performance

by
Juan Carlos López-Linares
1,2,*,
Alba Mei González-Galán
1,2,
Mónica Coca
1,2,
Susana Lucas
1,2 and
María Teresa García-Cubero
1,2
1
Department of Chemical Engineering and Environmental Technology, University of Valladolid, Dr. Mergelina s/n, 47011 Valladolid, Spain
2
Institute of Sustainable Processes, Department of Chemical Engineering and Environmental Technology, University of Valladolid, Dr. Mergelina s/n, 47011 Valladolid, Spain
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(14), 7808; https://doi.org/10.3390/app15147808
Submission received: 18 June 2025 / Revised: 9 July 2025 / Accepted: 10 July 2025 / Published: 11 July 2025
(This article belongs to the Section Applied Biosciences and Bioengineering)
Browse Figures
Versions Notes

Abstract

This study investigates the valorization of carrot discard, a carbohydrate-rich agricultural residue, for the production of 2,3-butanediol (2,3-BDO). The fermentation process was evaluated using two strains, Bacillus licheniformis DSM 8785 and Bacillus amyloliquefaciens DSM 7. Two process configurations were compared: separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). Additionally, to determine substrate and product inhibition thresholds, fermentation assays were conducted in semi-defined media with glucose concentrations ranging from 20 to 120 g/L. The SHF strategy proved more effective than the SSF configuration. Under the SHF configuration, B. amyloliquefaciens demonstrated superior performance, yielding 16.7 g/L of 2,3-BDO. In contrast, B. licheniformis was notable for its high capacity for acetoin synthesis, producing 24.2 g/L of acetoin in addition to 10.9 g/L of 2,3-BDO. Therefore, these findings demonstrate that carrot discard is a viable feedstock for the co-production of 2,3-BDO and acetoin.

1. Introduction

Carrots are one of the most significant root vegetables globally. In 2022, Spain’s production alone reached 387,907 t [1]. However, an estimated 25–30% of this harvest is discarded due to physical imperfections that render it unsuitable for the commercial market [2]. Carrot discards (CD) contain high levels of free sugars and structural carbohydrates [3,4], making them a potential feedstock for the production of biofuels and/or bioproducts (e.g., 2,3-butanediol (2,3-BDO)) using biological processes.
2,3-BDO is considered an important industrial platform, characterized to be green, natural, and sustainable. In addition, it is known as an interesting, valuable commercial chemical [5,6]. Its favorable physicochemical properties—including its colorless, odorless, and hygroscopic nature, high solubility in various solvents, and excellent biodegradability—underpin its use in numerous applications [7]. These include roles as a liquid fuel, cryoprotectant, fumigant, food additive, as carrier for pharmaceuticals, as intermediate for solvents and high-value-added products production, as cosmetic ingredient (i.e., as humectant, emollient, and antiseptic ingredient), and in the painting sector, among others [8,9].
Although 2,3-BDO is currently produced industrially by cracking petroleum-derived hydrocarbons, there is growing interest in its biological production from renewable, carbohydrate-rich residues. This bio-based route offers a path towards more sustainable, cost-effective, and large-scale synthesis [7]. In this way, according to Maina et al. [9], the 2,3-BDO global market could increase up to $220 million by 2027. A wide array of microorganisms have been identified as potent 2,3-BDO producers, including species of Enterobacter, Klebisella, Ralstonia, Serratia marcescens, Paenibacillus, and Bacillus, among others [7]. Among them, the 2,3-BDO production by Bacillus strains (such as B. licheniformis and B. amyloliquefaciens) is greatly recommended, since they are considered nonpathogenic (class 1) strains. Therefore, the 2,3-BDO industrial production using this type of strain will require of less strict safety regulations, and then, it could be cheaper and less complex [10,11]. Other than 2,3-BDO, Bacillus strains are able to produce considerable other by-products, such as acetoin, ethanol, and lactic, acetic, and citric acids, among others [12].
Acetoin is a naturally occurring flavor compound found in numerous foods, including fruits and fermented products like wine. As an important platform chemical, it is used extensively as a flavor and fragrance agent in the food and cosmetic industries, with global consumption reaching thousands of tons annually. Beyond these roles, acetoin serves as a crucial building block for synthesizing high-value chemicals, including optically active α-hydroxyketone derivatives, liquid crystal composites, and pharmaceuticals. It can also be catalytically converted into other valuable compounds such as diacetyl, acetoin acetate, and 2,3,5,6-tetramethylpyrazine [9,13]. Currently, industrial-scale acetoin production relies predominantly on chemical synthesis from fossil-based feedstocks. These processes often involve free-radical substitution reactions, which pose significant environmental concerns. While the direct cost of bio-based acetoin production may currently be higher than conventional chemical routes, the resulting product is considered “natural”. This classification makes it far more acceptable for use in the food, cosmetic, and pharmaceutical industries, where consumer preference and regulatory standards favor bio-derived ingredients. Consequently, the development of highly efficient and sustainable biological methods for acetoin production represents a significant research priority with substantial application value [13,14].
The conversion of complex carbohydrates in vegetable residues like CD into fermentable sugars requires enzymatic hydrolysis. This step can be performed prior to fermentation in a separate hydrolysis and fermentation (SHF) configuration. While SHF allows for the independent optimization of each process, an alternative approach is simultaneous saccharification and fermentation (SSF), where both stages occur in a single vessel. This integrated strategy can enhance energy efficiency, reduce capital costs, simplify scale-up, and mitigate challenges such as end-product inhibition and high medium viscosity [15,16].
Although the production of 2,3-BDO from carrot discards has been previously explored [3], significant gaps remain in process optimization, particularly regarding the use of industrially relevant, non-pathogenic bacteria and integrated bioprocessing strategies. This study, for the first time, investigates the co-production of 2,3-BDO and acetoin from untreated carrot discards using B. licheniformis and B. amyloliquefaciens. The efficacy of SHF and SSF configurations was systematically compared and, to further delineate the process boundaries, the substrate inhibition kinetics for both strains were analyzed. This work thus provides a comprehensive evaluation of novel bioprocessing routes for valorizing carrot waste into multiple value-added chemicals.

2. Materials and Methods

2.1. Raw Material

CD, carrot discarded due to physical imperfections that render it unsuitable for the commercial food market, were donated by a vegetable industry (Horcaol Cooperative Society, Olmedo, Valladolid, Spain). CD was milled (up to 1–3 mm particle size) with a household grinder (Taurus, Lérida, Spain) and stored at 4 °C until use. The composition was (% w/w dry matter) galacturonic acid, 11.2 ± 0.2; cellulose, 11.2 ± 0.1; hemicellulose, 5.5 ± 0.3; galactose + fructose in hemicellulose, 4.2 ± 0.2; arabinose in hemicellulose, 2.0 ± 0.2; acid-insoluble lignin (AIL), 0.3 ± 0.0; acid-soluble lignin (ASL), 1.6 ± 0.0; extractives, 58.8 ± 0.4; glucose in extractives, 15.3 ± 1.9; galactose + fructose in extractives, 12.6 ± 1.3; arabinose in extractives, 0.7 ± 0.3; ash, 7.5 ± 0.4; and acetyl groups, 0.6 ± 0.0 [3].

2.2. Enzymatic Hydrolysis of Carrot Discard

In order to obtain an enzymatic hydrolysate of CD for subsequent 2,3-BDO production, CD was subjected to enzymatic hydrolysis at 10% (w/v) substrate loading (25 g CD and 250 mL enzymatic solution), 50 °C, 150 rpm, and pH 4.8 for 24 h, using 1000 mL Erlenmeyer flasks and an orbital shaker (Optic Ivymen Systems, Comecta, Barcelona, Spain). Enzymatic solution was composed of a mixture of both Cellic CTec2 (10 FPU/g substrate) and Viscozyme L (10 FPU/g substrate) enzymes, from Novozymes A/S (Bagsværd, Denmark), using water as the solvent at pH 4.8. pH 4.8 was adjusted with 10 M NaOH solution. These conditions were used on the basis of previous results [3]. Once the enzymatic hydrolysis was finished, the resulting enzymatic hydrolysate was separated from residual CD by vacuum filtration, measured for its sugar content, and employed as fermentation broth for 2,3-BDO production.

2.3. Microorganism and Inoculum

Microorganisms used in this study were Bacillus licheniformis DSM 8785 and Bacillus amyloliquefaciens DSM 7, both from the microorganisms German collection (DSMZ, Leibniz, Germany). The reactivation of lyophilized cells was carried out in DSMZ liquid medium (at 30 °C and 150 rpm for 48 h), employing an orbital shaker (Optic Ivymen Systems, Comecta, Barcelona, Spain). The composition of DSMZ liquid medium was (g/L) peptone, 5; meat extract, 3; and MnSO4·H2O, 0.01, at pH 7. Once reactivated, both strains were stored as glycerol stock (40% (v/v)) at −80 °C until further use.
The inoculum cultivation (pre-culture) for both inocula was carried out in 250 mL Erlenmeyer flasks (at 37 °C and 150 rpm for 24 h, in a rotary shaker), using 50 mL of basal medium. The basal medium contained (per liter) 20 g glucose, 5 g yeast extract, 5 g Bacto tryptone, 7 g K2HPO4, 5.5 g KH2PO4, 1 g (NH4)2SO4, 0.25 g MgSO4·7H2O, 0.12 g Na2MoO4·2H2O, 0.021 g CaCl2·2H2O, 0.029 g Co(NO3)2·6H2O, 0.039 g Fe(NH4)2(SO4)2·6H2O, and 10 mL trace elements. The trace elements solution contained (per liter) 0.2 g nicotinic acid, 0.0262 g Na2SeO3·5H2O, 0.0037 g NiCl2·6H2O, 0.5 g MnCl2·4H2O, 0.1 g H3BO3, 0.0172 g AlK(SO4)2·12H2O, 0.001 g CuCl2·2H2O, and 0.554 g Na2EDTA·2H2O. All medium components were autoclaved (121 °C, 15 min), except the solutions containing Co(NO3)2 and Fe(NH4)2(SO4)2 as well as the trace elements solution, which were sterilized by filtration (using 0.2 μm cellulose nitrate filters) [17].
The inoculation was performed employing 1 mL of B. licheniformis or B. amyloliquefaciens glycerol stocks.

2.4. Fermentation Assays

2.4.1. Semi-Defined Fermentation Media

Semi-defined media, with different glucose concentrations (20, 40, 60, 80, 100, and 120 g/L), were used as fermentation broths for 2,3-BDO production. All semi-defined media were supplemented with the same nutrients employed in the pre-culture medium, and sterilized by filtration (using 0.2 μm cellulose nitrate filters).
Fermentation tests were performed at 37 °C, 150 rpm, 144 h, and pH 6.5 (without controlling through fermentation), using 250 mL Erlenmeyer flasks and 50 mL of fermentation broth per flask. The inoculum loading used was 4% (v/v) for B. licheniformis or B. amyloliquefaciens.

2.4.2. Separate Hydrolysis and Fermentation (SHF) of Carrot Discard

Enzymatic hydrolysate of CD (obtained in Section 2.2) was fermented to 2,3-BDO production by B. licheniformis or B. amyloliquefaciens. In this way, CD enzymatic hydrolysate was supplemented with the same nutrients employed in the pre-culture medium and pasteurized at 90 °C for 15 min. Fermentation experiments were carried out at 37 °C, 150 rpm, 144 h, and pH 6.5 (without controlling through fermentation), using 250 mL Erlenmeyer flasks and 50 mL of working volume. The inoculum loading used was 4% (v/v) for B. licheniformis or B. amyloliquefaciens.

2.4.3. Simultaneous Saccharification and Fermentation (SSF) of Carrot Discard

Ten percent (w/v) was the solid loading used in SSF experiments for 2,3-BDO production, and CD was the feedstock used. In this way, 5 g CD (dry matter) and 50 mL working solution were added to 250 mL Erlenmeyer flasks. The liquid phase contained the required nutrients (identical to the pre-culture medium), the enzymatic cocktail (Cellic CTec2 and Viscozyme L), and distilled water. Then, 50 mL is the total working volume, being its pH adjusted to 6.5 with 10 M NaOH solution. The volumes of Cellic CTec2 and Viscozyme L were calculated based on their respective activities (90 and 54.5 Filter Paper Units (FPU)/mL) to achieve an enzymatic loading of 10 FPU/g CD for each enzyme. Inoculum (B. licheniformis or B. amyloliquefaciens, 4% v/v) was also added. SSF tests were conducted at 37 °C, and 150 rpm for 144 h. In this way, at the beginning of the experiment (t = 0), the enzyme mixture (Cellic CTec2 and Viscozyme L) and B. licheniformis or B. amyloliquefaciens inocula were simultaneously added.
In all fermentation experiments, samples (0.3 mL) were withdrawn during fermentation (at t = 0, 2, 4, 6, 8, 10, 12, 22, 28, 32, 46, 56, and 72 h (and 144 h in the case of glucose semi-defined media by B. amyloliquefaciens, and all fermentations using carrot discard (both SHF and SSF))), centrifuged (at 13,500 rpm for 10 min), and analyzed for their contents in sugars, 2,3-BDO, ethanol, acetoin, pH, and cells. Fermentation experiments were conducted at least in duplicate.

2.5. Analytical Methods

High Performance Liquid Chromatography (HPLC) was used to analyze the content of sugars (glucose, galactose + fructose, and arabinose) and fermentation products (2,3-BDO, ethanol and acetoin), using a refractive index detector (Waters 2414, Milford, MA, USA), an Aminex HPX-87H column (Bio-Rad, Alcobendas, Madrid) (at 60 °C), and 0.01 N H2SO4 (0.6 mL/min) as the mobile phase. On the other hand, cell concentration in the fermentation tests was monitored by measuring the optical density (OD) at 600 nm using a Uvmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan).
All analytical determinations were carried out in triplicate, and the average results are shown.

2.6. Data Analysis

The statistical differences were determined using variance analysis (ANOVA) at a confidence level of 95% (p < 0.05). The Tukey multiple range test was used to find significantly different means.

3. Results and Discussion

3.1. 2,3-BDO Production from Glucose Semi-Defined Media

3.1.1. 2,3-BDO Fermentation by B. licheniformis

To investigate the substrate and product inhibition thresholds for 2,3-BDO production by B. licheniformis, fermentation assays were conducted in a semi-defined medium with initial glucose concentrations ranging from 20 to 120 g/L. The results of these experiments are summarized in Table 1 and Figure 1.
Regarding substrate consumption, B. licheniformis fully metabolized the available glucose by the end of fermentation (t = 72 h) (Figure 1a). As expected, the rate of glucose consumption was inversely related to its initial concentration. For instance, in cultures initiated with 20 g/L glucose (G20), the substrate was depleted within 12 h. In contrast, complete consumption required up to 72 h in fermentations starting with 120 g/L glucose (G120).
As illustrated in Table 1 and Figure 1b, final 2,3-BDO production correlated positively with the initial glucose concentration, reaching a maximum of 21.8 g/L at 120 g/L glucose. Furthermore, at initial glucose concentrations up to 80 g/L, peak 2,3-BDO production was achieved in relatively short fermentation times (t < 32 h), a finding with positive implications for process economics. Notably, high glucose uptake (>85%) occurred in all cultures by the time maximum 2,3-BDO production were reached (Table 1).
While 2,3-BDO yields (from 0.12 to 0.20 g/g) and productivities (from 0.201 to 0.391 g/L∙h) rose with initial glucose concentrations up to 100 g/L, concentrations exceeding this value led to a decline in both parameters (Table 1), probably due to partial inhibition of B. licheniformis by high concentration of substrate and/or product. A similar trend was observed for cell growth (Table 1). pH values were also measured during fermentation (Figure S1a). As can be observed, in all cases, pH decreased from 6.6 up to about 5.5 during the first hours of fermentation (10–12 h), being increased again after this fermentation time (up to 6.9, for example, for G20 at the end of fermentation).
In addition to 2,3-BDO, the fermentation yielded by-products such as ethanol and acetoin [18]. Both compounds originate from pyruvate, which is considered as an important intermediate in 2,3-BDO fermentation process; specially, ethanol is produced through successive pyruvate–formate lyase, acetaldehyde dehydrogenase, and ethanol dehydrogenase pathways; while acetoin is generated by successive α-acetolactate synthase and 4,α-acetolactate decarboxylase pathways [18]. While only low concentrations of ethanol were detected (≤1.1 g/L), significant titers of acetoin (5.3–32.5 g/L) were produced across all conditions, with the maximum value observed at 120 g/L initial glucose (Table 1, Figure 1c,d). This high acetoin co-production is noteworthy given its value as a platform chemical in numerous sectors, including the food, cosmetic, pharmaceutical, and agricultural industries [9]. Moreover, acetic and lactic acids can also be originated in 2,3-BDO fermentations by B. licheniformis [12]. In this way, notable concentrations of lactic acid (1.8–4.2 g/L) (Figure S2a) and lower amounts of acetic acid (0.1–0.6 g/L) (Figure S2b) were also detected in this work at the time of maximum 2,3-BDO production.
Furthermore, it is worth mentioning that once glucose levels in the medium were depleted, B. licheniformis began to consume the previously produced 2,3-BDO (Figure 1a,b). This phenomenon is directly linked to the cell’s need to maintain redox balance and generate energy under nutrient-limiting conditions. The synthesis of 2,3-BDO is an NADH-dependent reduction of acetoin. However, when levels of carbohydrates in the fermentation medium are low, 2,3-BDO can be reversibly turned into acetoin, regenerating the NADH and then keeping a continual oxidation–reduction state. When the sugar is depleted from the medium, NADH generation is decreased or stopped, and hence, 2,3-BDO is oxidized to acetoin, leading to a replenishment of NADH for cellular function [9]. Moreover, according to Sonenshein et al. [19], when glucose has been fully consumed or its levels are low, sporulation of the strain Bacillus is triggered, and cells reintroduce the products generated (i.e., 2,3-BDO), metabolizing them to generate additional energy. This behavior—the re-consumption of 2,3-BDO upon substrate depletion—is well-documented in the literature for various Bacillus and related species under different fermentation conditions: for example, in the 2,3-BDO and acetoin generation from glucose fermentation broth by Bacillus amyloliquefaciens 83 [20]; in the 2,3-BDO fermentation of sorghum stalk biomass using Bacillus licheniformis DSM 8785 [21]; in the 2,3-BDO production from carrot discard by Paenibacillus polymyxa DSM 365 [3]. This behavior was not appreciated for fermentation with 120 g/L initial glucose (G120), probably due to the considerable glucose levels remaining in fermentation broth along process.
To mitigate this product loss and maximize the final 2,3-BDO titer, a fed-batch fermentation strategy could be implemented. This approach would involve the controlled addition of a sugar solution to maintain a minimum substrate concentration, thereby preventing the metabolic shift towards 2,3-BDO oxidation. For instance, Guragain et al. [21] suggest maintaining glucose levels above 10 g/L to ensure continuous 2,3-BDO accumulation and avoid its conversion back to acetoin, which is critical for simplifying downstream processing and improving process economics.

3.1.2. 2,3-BDO Fermentation by B. amyloliquefaciens

To provide a comparative analysis, fermentation assays were conducted with B. amyloliquefaciens under the same conditions used for B. licheniformis, with initial glucose concentrations ranging from 20 to 120 g/L. The results are presented in Table 2 and Figure 2.
B. amyloliquefaciens achieved high glucose consumption (≥90%) by the end of fermentation (t = 144 h) in cultures with initial concentrations up to 80 g/L (Table 2). However, a further increase in the initial glucose concentration resulted in substantial residual substrate, with up to 60% remaining unconsumed in the 120 g/L culture (Figure 2a). Correspondingly, the time required for maximum substrate utilization increased with the initial glucose load, reaching up to 56 h for the G20–G80 cultures.
Maximum 2,3-BDO concentrations rose from 5.8 to 19.8 g/L as the initial glucose concentration was increased from 20 to 100 g/L (Table 2, Figure 2b). However, increasing the initial concentration to 120 g/L resulted in a 24.2% decrease in the final 2,3-BDO production. This suggests a strong substrate inhibition effect on B. amyloliquefaciens at glucose concentrations exceeding 100 g/L. Notably, after glucose depletion, the produced 2,3-BDO was subsequently re-consumed by the microorganism in all tested conditions (Figure 2b).
The 2,3-BDO yields achieved by B. amyloliquefaciens were consistently high, ranging from 0.29 to 0.39 g/g (Table 2). Notably, the maximum yield (0.39 g/g) occurred at the highest initial glucose concentration (120 g/L). In contrast, volumetric productivity (0.265–0.348 g/L∙h) peaked at 40 g/L initial glucose and decreased at higher concentrations. This decline is attributable to the longer fermentation times needed to achieve peak production under substrate stress (Table 2). Cell growth followed a similar trend to productivity. pH was also analyzed during fermentation (Figure S1b). As can be seen, independently of initial glucose concentration, pH decreased from 6.6 up to about 5.5 during the first hours of fermentation (12 h). However, after 12 h fermentation, pH increased up to 6.5–6.8 g/L (at the end of fermentation) for G20 and G40, while it decreased (up to 4.7–5.2) for G ≥ 60.
A key distinction from B. licheniformis was the absence of ethanol production by B. amyloliquefaciens. Nevertheless, considerable acetoin concentrations were detected (3.3–10.3 g/L), peaking at an initial glucose concentration of 100 g/L (Table 2, Figure 2c). Additionally, low concentrations of lactic acid (0.5–1.8 g/L) (Figure S3a) and acetic acid (1.3–1.7 g/L) (Figure S3b) were quantified at the time of maximum 2,3-BDO production, which is consistent with previous findings for B. amyloliquefaciens [22]. This by-product profile, characterized by the absence of ethanol, enhances the potential of this process within an integrated biorefinery concept by simplifying downstream separation.

3.1.3. Comparison of 2,3-BDO Fermentation by B. licheniformis and B. amyloliquefaciens

A direct comparison of the two strains revealed key differences in substrate utilization and fermentation kinetics. B. licheniformis achieved complete glucose consumption across all tested initial concentrations (20–120 g/L), whereas B. amyloliquefaciens demonstrated efficient uptake (≥90%) only at concentrations up to 80 g/L (Table 1 and Table 2, Figure 1 and Figure 2). Furthermore, B. licheniformis exhibited faster overall kinetics, completing fermentation in 72 h compared to 144 h for B. amyloliquefaciens (Figure 1a and Figure 2a).
At initial glucose concentrations between 20 and 80 g/L, B. amyloliquefaciens produced significantly higher 2,3-BDO titers (p < 0.05), yielding 1.4- to 2.4-fold more product than B. licheniformis (Table 1 and Table 2, Figure 1b and Figure 2b). However, this advantage was lost at concentrations above 80 g/L. At 100 g/L initial glucose, the 2,3-BDO production from both strains was comparable (18.0–19.8 g/L), while at 120 g/L, B. amyloliquefaciens produced substantially less 2,3-BDO than B. licheniformis (15.0 g/L vs. 21.8 g/L). These results indicate that B. amyloliquefaciens is more susceptible to substrate inhibition at high glucose loads (>80 g/L) than B. licheniformis.
Regarding process efficiency, B. amyloliquefaciens demonstrated significantly higher 2,3-BDO yields (p < 0.05), ranging from 0.29 to 0.39 g/g compared to 0.12–0.20 g/g for B. licheniformis (Table 1 and Table 2). The yield from B. amyloliquefaciens reached up to 78% of the theoretical maximum (0.5 g/g) [23]. In contrast, B. licheniformis consistently reached its peak titer more rapidly (e.g., t = 28 h vs. 56 h at 60 g/L glucose). Consequently, despite the yield disparity, the overall volumetric productivities of the two strains were not significantly different (p > 0.05), with ranges of 0.201–0.391 g/L∙h for B. licheniformis and 0.265–0.348 g/L∙h for B. amyloliquefaciens. Similarly, final cell densities were comparable between the strains (OD600 6.5–16.2 vs. 8.0–14.4) (Table 1 and Table 2). Considering pH parameter, although it decreased during the first 10–12 h of fermentation for both microorganisms, after this fermentation time, for G ≥ 60 g/L initial glucose, B. licheniformis increased the pH of medium, while a decrease in pH was observed in fermentations by B. amyloliquefaciens (Figure S1a,b).
The two strains also exhibited distinct by-product profiles. B. licheniformis was a more prolific producer of acetoin, generating significantly higher concentrations (5.3–32.5 g/L) than B. amyloliquefaciens (3.3–10.3 g/L) (Table 1 and Table 2, Figure 1c and Figure 2c). Crucially, the production dynamics differed: B. licheniformis co-produced acetoin and 2,3-BDO from the onset of fermentation, whereas acetoin synthesis by B. amyloliquefaciens was delayed, increasing only as glucose was depleted and 2,3-BDO levels rose. Consistent with our results, B. licheniformis has been previously recognized for its capacity to co-produce significant amounts of acetoin alongside 2,3-BDO [21]. In contrast, various B. amyloliquefaciens strains are often cited for their high efficiency in selectively producing 2,3-BDO. In this way, for example, Yang et al. [22] reported that B. amyloliquefaciens produced high titers of 2,3-BDO (20.6–40.7 g/L) from spirit-based distillers’ grain, with comparatively low acetoin co-production (2.9–5.4 g/L). This metabolic bifurcation towards either acetoin or 2,3-BDO is regulated by several factors, most notably the intracellular redox state (NAD+/NADH ratio) and the concentration of dissolved oxygen in the medium [24]. The final conversion step, the reduction of acetoin to 2,3-BDO, is NADH-dependent. Therefore, the direction of this reversible reaction is highly sensitive to environmental conditions. Specifically, dissolved oxygen concentrations below a critical threshold (typically <100 ppb) favor the regeneration of NAD+ via 2,3-BDO formation, whereas higher oxygen levels tend to result in the accumulation of acetoin [25]. Consequently, the inherent differences in oxygen consumption rates and redox management between the B. licheniformis and B. amyloliquefaciens strains likely underlie the divergent product profiles observed in this work. Furthermore, B. licheniformis produced low levels of ethanol (0.5–1.1 g/L) (Table 1, Figure 1d), a by-product that was entirely absent in B. amyloliquefaciens fermentations.

3.2. Comparative Analysis of SHF and SSF Configurations for 2,3-BDO Production from Carrot Discard

To assess the feasibility of using carrot discard (CD) as a feedstock, two process configurations—separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF)—were evaluated for both B. licheniformis and B. amyloliquefaciens. For the SHF process, an initial enzymatic hydrolysis of the CD yielded a sugar-rich hydrolysate with a total sugar content of 67.7 g/L. The composition was as follows (in g/L): glucose, 41.5; fructose and galactose, 24.2; arabinose, 2.0; formic acid, 0.3; and acetic acid, 0.7. In contrast, the SSF strategy involved conducting the hydrolysis and fermentation steps concurrently in a single vessel. The performance of both strains under each configuration is detailed in Table 3 and Figure 3.
Under the SHF configuration, B. licheniformis completely consumed all available sugars from the CD hydrolysate within 46 h (Figure 3a), showing a preference for glucose (depleted in 28 h) over fructose, galactose, and arabinose (depleted in 46 h) (Figure S4). This phenomenon is characteristic of catabolite repression, a well-documented regulatory mechanism in many bacteria. In the presence of a preferred carbon source like glucose, the expression and activity of enzymes required for the transport and metabolism of secondary sugars are repressed [26]. For example, the metabolism of pentoses such as xylose is often inhibited by glucose through the repression of key enzymes like xylose reductase and xylitol dehydrogenase. Only after glucose has been substantially depleted from the medium are these enzymatic pathways derepressed, allowing for the consumption of the remaining sugars. This pattern of sequential sugar utilization is consistent with numerous studies on 2,3-BDO production from mixed-sugar feedstocks. Guragain et al. [21] observed a much higher glucose uptake rate compared to pentose uptake by B. licheniformis DSM 8785 during fermentation of sorghum stalk biomass. Similarly, López-Linares et al. [3] found that while Paenibacillus polymyxa DSM 365 could co-utilize glucose and fructose to some extent, galactose consumption was significantly delayed until glucose was nearly exhausted, being the glucose utilization 10.7 times greater than that of galactose. In other work, diauxic growth patterns have also been clearly demonstrated during the co-fermentation of glucose and pentoses [5]. The results of our study thus align perfectly with these established metabolic behaviors.
This resulted in a final 2,3-BDO production of 10.9 g/L, a yield of 0.17 g/g, and a productivity of 0.236 g/L∙h (Table 3, Figure 3b). Notably, this strain co-produced a substantial acetoin titer of 24.2 g/L (Table 3, Figure 3c), alongside lower concentrations of ethanol (0.4 g/L) (Table 3, Figure 3d), acetic acid (2.2 g/L) (Figure S5b), and lactic acid (2.5 g/L) (Figure S5a). In stark contrast, the SSF strategy was ineffective. Sugar metabolism was delayed by 6–8 h and ultimately stalled, resulting in only 51% sugar uptake after 144 h (Figure 3a). Consequently, 2,3-BDO production was minimal (0.9 g/L). This poor performance is likely attributable to cellular stress from the high solid load and low water content, as well as osmotic pressure in the SSF environment, which is known to inhibit bacterial growth and metabolic function [27]. In this way, according to Verardi et al. [28], the high solid content in the process could result in deteriorated enzyme and microorganism activities and an increase in viscosity, hindering the homogeneous and effective distribution of the enzymes and microorganisms. Moreover, in the SSF configuration, the fermentation began with a heterogeneous slurry where sugars were not immediately bioavailable. Then, this fact could also interfere negatively with the metabolism of both Bacillus strains. The metabolic response to these challenging conditions was evident in our results. For both B. licheniformis and B. amyloliquefaciens, the metabolic flux during SSF shifted decisively away from 2,3-BDO production and towards the synthesis of organic acids, resulting in high titers of lactic acid (11.1–12.5 g/L) and acetic acid (5.1–5.3 g/L) (Figure S5). This shift towards organic acid production under stressful SSF conditions is consistent with findings in the literature, where SSF processes have been effectively employed to produce lactic acid from corn crop residues by Bacillus coagulans [29] and succinic acid from corn stover using Actinobacillus succinogenes [30]. Therefore, the SHF configuration was unequivocally superior for B. licheniformis.
Similarly, when using B. amyloliquefaciens in the SHF configuration, all sugars were consumed by the end of fermentation (t = 144 h), with over 80% uptake achieved by 56 h (Figure 3a). This strain also preferentially consumed glucose (72 h) over other sugars (144 h) (Figure S4). The SHF process yielded a high 2,3-BDO production of 16.7 g/L within 56 h, corresponding to a yield of 0.33 g/g and a productivity of 0.298 g/L∙h (Table 3, Figure 3b). Co-produced acetoin reached 8.0 g/L (Table 3, Figure 3c), while ethanol was minimal (0.2 g/L) (Table 3, Figure 3d) and organic acids were low (3.1–3.4 g/L) (Figure S5). As observed with the other strain, the SSF configuration was markedly less efficient for B. amyloliquefaciens. Only 46.4% of the available sugar was consumed, yielding just 4.1 g/L of 2,3-BDO and 3.1 g/L of acetoin (Table 3 and Figure 3). Again, metabolism shifted towards organic acids (12.5 g/L lactic acid and 5.3 g/L acetic acid) (Figure S5), indicating process inhibition [27]. Thus, SHF was also the best strategy for this strain.
The behavior of pH was also very different for SHF and SSF configurations (Figure S1c), in both fermentations by B. licheniformis and B. amyloliquefaciens. While pH decreased (up to 4.2–4.4) during the whole fermentation in SSF, probably due to the formation of lactic and acetic acids, in SHF, a decrease was observed during the first hours of the process (up to 5.6–5.7 at 22 h), increasing after this fermentation time.
In a direct comparison using the SHF configuration, B. amyloliquefaciens was the more effective 2,3-BDO producer, achieving a significantly higher concentration (16.7 vs. 10.9 g/L), yield (0.33 vs. 0.17 g/g), and productivity (0.298 vs. 0.236 g/L∙h) than B. licheniformis (p < 0.05). In contrast, B. licheniformis demonstrated a remarkable capacity for acetoin synthesis, producing a 3-fold higher concentration than B. amyloliquefaciens (24.2 vs. 8.0 g/L). These divergent product profiles on CD align with the findings from the semi-defined media (Section 3.1). A crucial finding for process viability is that these results were achieved using untreated CD, circumventing the need for costly pretreatment and thereby enhancing the potential profitability of this valorization route.
The optimal results obtained in this study—notably a 2,3-BDO titer of 16.7 g/L, a yield of 0.33 g/g, and a productivity of 0.298 g/L∙h—are highly competitive when contextualized with the existing literature. Lower 2,3-BDO levels (5.2–5.9 g/L) were achieved by OHair et al. [31] from aqueous solutions of pepper, pineapple, and cabbage waste, using B. licheniformis YNP5-TSU. Bacillus strain (B. subtilis LOCK 1086) was also employed in the fermentation of apple pomace enzymatic hydrolysates, resulting in lower 2,3-BDO concentrations (12.8 g/L) than those reported in this work [32]. Not very different 2,3-BDO production (18.8 g/L), but much lower acetoin levels (2.1 g/L) were also reported from carrot discard enzymatic hydrolysate, but using P. polymyxa DSM 365 [3]. Sikora et al. [33] also stated similar or even much lower 2,3-BDO concentrations (16.5, 10.7, and 5 g/L) than those found in this study from enzymatic hydrolysates of apple pomace, dried sugar beet pulp, and potato pulp, respectively, by B. amyloliquefaciens TUL 308. López-Linares et al. [34] obtained much lower 2,3-BDO concentration, yield, and productivity (3.7 g/L, 0.14 g/g, and 0.10 g/L⋅h, respectively) than those achieved in this study from brewer’s spent grain in the co-culture fermentation by Paenibacillus polymyxa DSM 365 and Rhodococcus sp. B. licheniformis NCIMB 8059 was also used for 2,3-BDO production from enzymatic apple pomace hydrolysate, yielding only 11.6 g/L 2,3-BDO [35]. Slightly lower 2,3-BDO yields (0.29–0.30 g/g) than those achieved in this work were reported by Yang et al. [22] from spirit-based distillers’ grain (SDG) by B. amyloliquefaciens B10-127.

4. Conclusions

This study successfully demonstrates that carrot discard is a viable feedstock for the co-production of 2,3-butanediol and acetoin using non-pathogenic Bacillus strains. The separate hydrolysis and fermentation (SHF) configuration proved unequivocally superior to the simultaneous saccharification and fermentation (SSF) strategy for both strains, mitigating the inhibitory effects observed. A key finding is the distinct product specialization of the two strains: B. amyloliquefaciens emerged as the superior producer of 2,3-BDO, while B. licheniformis showed a remarkable capacity for acetoin synthesis. Under the optimal SHF process, B. amyloliquefaciens yielded 16.7 g of 2,3-BDO and 8.0 g of acetoin per 100 g of dry carrot discard. In contrast, B. licheniformis produced 10.9 g of 2,3-BDO and a substantial 24.2 g of acetoin under the same conditions. Future research should focus on overcoming the limitations of the SSF process, potentially through fed-batch strategies, to alleviate the inhibition caused by high osmotic pressure and thereby develop a more integrated and cost-effective single-vessel bioprocess.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app15147808/s1, Figure S1: Fermentation kinetics (pH) in: semi-defined media of glucose by B. licheniformis (a); semi-defined media of glucose by B. amyloliquefaciens (b); carrot discard fermentations in SHF and SSF configurations by B. licheniformis (continuous lines) and B. amyloliquefaciens (dashed lines) (c); Figure S2: Fermentation kinetics in semi-defined media of glucose by B. licheniformis: lactic acid (a) and acetic acid (b); Figure S3: Fermentation kinetics in semi-defined media of glucose by B. amyloliquefaciens: lactic acid (a) and acetic acid (b); Figure S4: Kinetics of carrot discard fermentations in SHF and SSF configurations by B. licheniformis (continuous lines) and B. amyloliquefaciens (dashed lines): consumption of glucose (a), fructose + galactose (b), and arabinose (c); Figure S5: Kinetics of carrot discard fermentations in SHF and SSF configurations by B. licheniformis (continuous lines) and B. amyloliquefaciens (dashed lines): lactic acid (a) and acetic acid (b).

Author Contributions

Conceptualization, M.T.G.-C., M.C., S.L., and J.C.L.-L.; methodology, J.C.L.-L. and A.M.G.-G.; formal analysis, M.C. and S.L.; investigation, J.C.L.-L. and A.M.G.-G.; resources, J.C.L.-L.; writing—original draft preparation, J.C.L.-L.; writing—review and editing, M.T.G.-C. and J.C.L.-L.; supervision, M.T.G.-C., M.C., S.L., and J.C.L.-L.; project administration, M.T.G.-C. and J.C.L.-L.; funding acquisition, M.T.G.-C. and J.C.L.-L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Spanish Ministry of Science and Innovation (project PID2020-115110RB-I00/AEI/10.13039/501100011033 and project PID2023-147967OB-I00/MCIU/AEI/10.13039/501100011033/FEDER, EU) and by the Junta de Castilla y León (UIC 320).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Acknowledgments

Juan Carlos López-Linares gratefully acknowledges the research grant received from the University of Valladolid (UVa postdoctoral). The authors would like to thank the company Horcaol for supplying the carrot discards.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. MAPA Ministry of Agriculture, Fisheries and Food. “Anuario de Estadística”. Available online: https://www.mapa.gob.es/es/estadistica/temas/publicaciones/anuario-de-estadistica/default.aspx (accessed on 28 May 2025).
  2. Encalada, A.M.I.; Pérez, C.D.; Flores, S.K.; Rossetti, L.; Fissore, E.N.; Rojas, A.M. Antioxidant pectin enriched fractions obtained from discarded carrots (Daucus carota L.) by ultrasound-enzyme assisted extraction. Food Chem. 2019, 289, 453–460. [Google Scholar] [CrossRef]
  3. López-Linares, J.C.; Mateo Martínez, A.; Coca, M.; Lucas, S.; García-Cubero, M.T. Carrot discard as a promising feedstock to produce 2,3-butanediol by fermentation with P. polymyxa DSM 365. Bioengineering 2023, 10, 937. [Google Scholar] [CrossRef] [PubMed]
  4. Ramos-Andrés, M.; Aguilera-Torre, B.; García-Serna, J. Hydrothermal production of high-molecular weight hemicellulose-pectin, free sugars and residual cellulose pulp from discarded carrots. J. Clean. Prod. 2021, 290, 125179. [Google Scholar] [CrossRef]
  5. Okonkwo, C.C.; Ujor, V.; Ezeji, T.C. Production of 2,3-Butanediol from non-detoxified wheat straw hydrolysate: Impact of microbial inhibitors on Paenibacillus polymyxa DSM 365. Ind. Crops Prod. 2021, 159, 113047. [Google Scholar] [CrossRef]
  6. Hong, E.; Kim, D.; Kim, J.; Kim, J.; Yoon, S.; Rhie, S.; Ha, S.; Ryu, Y. Optimization of alkaline pretreatment on corn stover for enhanced production of 1.3-propanediol and 2,3-butanediol by Klebsiella pneumoniae AJ4. Biomass Bioenergy 2015, 77, 177–185. [Google Scholar] [CrossRef]
  7. Xie, S.; Li, Z.; Zhu, G.; Song, W.; Yi, C. Cleaner production and downstream processing of bio-based 2,3-butanediol: A review. J. Clean. Prod. 2022, 343, 131033. [Google Scholar] [CrossRef]
  8. Hazeena, S.H.; Nair Salini, C.; Sindhu, R.; Pandey, A.; Binod, P. Simultaneous saccharification and fermentation of oil palm front for the production of 2,3-butanediol. Bioresour. Technol. 2019, 278, 145–149. [Google Scholar] [CrossRef]
  9. Maina, S.; Prabhu, A.A.; Vivek, N.; Vlysidis, A.; Koutinas, A.; Kumar, V. Prospects on bio-based 2,3-butanediol and acetoin production: Recent progress and advances. Biotechnol. Adv. 2022, 54, 107783. [Google Scholar] [CrossRef]
  10. Yang, Z.; Zhang, Z. Recent advances on production of 2, 3-butanediol using engineered microbes. Biotechnol. Adv. 2019, 37, 569–578. [Google Scholar] [CrossRef]
  11. Jiang, L.-Q.; Fang, Z.; Zhao, Z.-L.; He, F.; Li, H.-B. 2,3-butanediol and acetoin production from enzymatic hydrolysate of ionic liquid-pretreated cellulose by Paenibacillus polymyxa. BioResources 2015, 10, 1318–1329. [Google Scholar] [CrossRef]
  12. Xu, Y.; Zhuang, J.; Wang, S.; Dai, J.; Xiu, Z.L. Integration of 2,3-butanediol production and extraction of active components from Chinese herbs by Bacillus licheniformis and evaluation of fermentation products in vitro. Process Biochem. 2025, 148, 150–156. [Google Scholar] [CrossRef]
  13. Meng, W.; Ma, C.; Xu, P.; Gao, C. Biotechnological production of chiral acetoin. Trends Biotechnol. 2022, 40, 958–973. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, Q.; Bao, T.; Hu, M.; Xu, M.; Rao, Z.; Zhang, X. Efficient acetoin production in Bacillus subtilis by multivariate modular metabolic engineering with spatiotemporal modulation. ACS Sustain. Chem. Eng. 2025, 13, 1927–1936. [Google Scholar] [CrossRef]
  15. Olofsson, K.; Bertilsson, M.; Lidén, G. A short review on SSF—An interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnol. Biofuels 2008, 1, 7. [Google Scholar] [CrossRef]
  16. Qiu, W.y.; Dong, C.; Guo, J.j.; Xia, B.; Xu, L.; Fang, Z. Enhancing xylose fermentation to maximize net energy gain of lime-pretreated wheat straw by delayed fed-batch simultaneous saccharification and fermentation. Biomass Bioenergy 2025, 198, 107865. [Google Scholar] [CrossRef]
  17. Jurchescu, I.M.; Hamann, J.; Zhou, X.; Ortmann, T.; Kuenz, A.; Prüße, U.; Lang, S. Enhanced 2,3-butanediol production in fed-batch cultures of free and immobilized Bacillus licheniformis DSM 8785. Appl. Microbiol. Biotechnol. 2013, 97, 6715–6723. [Google Scholar] [CrossRef] [PubMed]
  18. Hakizimana, O.; Matabaro, E.; Lee, B.H. The current strategies and parameters for the enhanced microbial production of 2,3-butanediol. Biotechnol. Rep. 2020, 25, e00397. [Google Scholar] [CrossRef]
  19. Sonenshein, A.L. Control of key metabolic intersections in Bacillus subtilis. Nat. Rev. Microbiol. 2007, 5, 917–927. [Google Scholar] [CrossRef]
  20. Cristiano-Fajardo, S.A.; Flores, C.; Flores, N.; Tinoco-Valencia, R.; Serrano-Carreón, L.; Galindo, E. Glucose limitation and glucose uptake rate determines metabolite production and sporulation in high cell density continuous cultures of Bacillus amyloliquefaciens 83. J. Biotechnol. 2019, 299, 57–65. [Google Scholar] [CrossRef]
  21. Guragain, Y.N.; Chitta, D.; Karanjikar, M.; Vadlani, P.V. Appropriate lignocellulosic biomass processing strategies for efficient 2,3-butanediol production from biomass-derived sugars using Bacillus licheniformis DSM 8785. Food Bioprod. Process. 2017, 104, 147–158. [Google Scholar] [CrossRef]
  22. Yang, T.; Rao, Z.; Zhang, X.; Xu, M.; Xu, Z.; Yang, S.T. Economic conversion of spirit-based distillers’ grain to 2,3-butanediol by Bacillus amyloliquefaciens. Process Biochem. 2015, 50, 20–23. [Google Scholar] [CrossRef]
  23. Qin, J.; Xiao, Z.; Ma, C.; Xie, N.; Liu, P.; Xu, P. Production of 2,3-Butanediol by Klebsiella pneumoniae using glucose and ammonium phosphate. Chin. J. Chem. Eng. 2006, 14, 132–136. [Google Scholar] [CrossRef]
  24. Celińska, E.; Grajek, W. Biotechnological production of 2,3-butanediol—Current state and prospects. Biotechnol. Adv. 2009, 27, 715–725. [Google Scholar] [CrossRef] [PubMed]
  25. Moes, J.; Griot, M.; Keller, J.; Heinzle, E.; Dunn, I.J.; Bourne, J.R. A microbial culture with oxygen-sensitive product distribution as a potential tool for characterizing bioreactor oxygen transport. Biotechnol. Bioeng. 1985, 27, 482–489. [Google Scholar] [CrossRef]
  26. Slininger, P.J.; Thompson, S.R.; Weber, S.; Liu, Z.L.; Moon, J. Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag. Biotechnol. Bioeng. 2011, 108, 1801–1815. [Google Scholar] [CrossRef]
  27. Jørgensen, H.; Vibe-Pedersen, J.; Larsen, J.; Felby, C. Liquefaction of lignocellulose at high-solids concentrations. Biotechnol. Bioeng. 2007, 96, 862–870. [Google Scholar] [CrossRef]
  28. Verardi, A.; Sangiorgio, P.; Blasi, A.; Lopresto, C.G.; Calabrò, V. Bioconversion of crop residues using alternative fermentation-based approaches. Front. Biosci. Elit. 2023, 15, 17. [Google Scholar] [CrossRef]
  29. Malacara-Becerra, A.; Melchor-Martínez, E.M.; Sosa-Hernández, J.E.; Riquelme-Jiménez, L.M.; Mansouri, S.S.; Iqbal, H.M.N.; Parra-Saldívar, R. Bioconversion of corn crop residues: Lactic acid production through simultaneous saccharification and fermentation. Sustainability 2022, 14, 11799. [Google Scholar] [CrossRef]
  30. Zheng, P.; Fang, L.; Xu, Y.; Dong, J.J.; Ni, Y.; Sun, Z.H. Succinic acid production from corn stover by simultaneous saccharification and fermentation using Actinobacillus succinogenes. Bioresour. Technol. 2010, 101, 7889–7894. [Google Scholar] [CrossRef]
  31. OHair, J.; Jin, Q.; Yu, D.; Wu, J.; Wang, H.; Zhou, S.; Huang, H. Non-sterile fermentation of food waste using thermophilic and alkaliphilic Bacillus licheniformis YNP5-TSU for 2,3-butanediol production. Waste Manag. 2021, 120, 248–256. [Google Scholar] [CrossRef]
  32. Białkowska, A.M.; Jędrzejczak-Krzepkowska, M.; Gromek, E.; Krysiak, J.; Sikora, B.; Kalinowska, H.; Kubik, C.; Schütt, F.; Turkiewicz, M. Effects of genetic modifications and fermentation conditions on 2,3-butanediol production by alkaliphilic Bacillus subtilis. Appl. Microbiol. Biotechnol. 2016, 100, 2663–2676. [Google Scholar] [CrossRef] [PubMed]
  33. Sikora, B.; Kubik, C.; Kalinowska, H.; Gromek, E.; Białkowska, A.; Jędrzejczak-Krzepkowska, M.; Schüett, F.; Turkiewicz, M. Application of byproducts from food processing for production of 2,3-butanediol using Bacillus amyloliquefaciens TUL 308. Prep. Biochem. Biotechnol. 2016, 46, 610–619. [Google Scholar] [CrossRef] [PubMed]
  34. López-Linares, J.C.; Rama, E.; García-Cubero, M.T.; Coca, M.; Perez, C.L.; Yamakawa, C.K.; Dragone, G.; Mussatto, S.I. Enhancing 2,3-butanediol and acetoin production from brewer’s spent grain hemicellulosic hydrolysate through bacterial co-cultivation. New Biotechnol. 2025, 88, 22–31. [Google Scholar] [CrossRef] [PubMed]
  35. Białkowska, A.M.; Gromek, E.; Krysiak, J.; Sikora, B.; Kalinowska, H.; Jędrzejczak-Krzepkowska, M.; Kubik, C.; Lang, S.; Schütt, F.; Turkiewicz, M. Application of enzymatic apple pomace hydrolysate to production of 2,3-butanediol by alkaliphilic Bacillus licheniformis NCIMB 8059. J. Ind. Microbiol. Biotechnol. 2015, 42, 1609–1621. [Google Scholar] [CrossRef]
Applsci 15 07808 g001aApplsci 15 07808 g001b
Figure 1. Fermentation kinetics in semi-defined media of glucose by B. licheniformis. Glucose consumption (a), and production of 2,3-butanediol (b), acetoin (c) and ethanol (d).
Figure 1. Fermentation kinetics in semi-defined media of glucose by B. licheniformis. Glucose consumption (a), and production of 2,3-butanediol (b), acetoin (c) and ethanol (d).
Applsci 15 07808 g001aApplsci 15 07808 g001b
Applsci 15 07808 g002
Figure 2. Fermentation kinetics in semi-defined media of glucose by B. amyloliquefaciens. Glucose consumption (a), and production of 2,3-butanediol (b) and acetoin (c).
Figure 2. Fermentation kinetics in semi-defined media of glucose by B. amyloliquefaciens. Glucose consumption (a), and production of 2,3-butanediol (b) and acetoin (c).
Applsci 15 07808 g002
Applsci 15 07808 g003aApplsci 15 07808 g003b
Figure 3. Kinetics of carrot discard fermentations in SHF and SSF configurations by B. licheniformis (continuous lines) and B. amyloliquefaciens (dashed lines). Glucose consumption (a), and production of 2,3-butanediol (b), acetoin (c), and ethanol (d).
Figure 3. Kinetics of carrot discard fermentations in SHF and SSF configurations by B. licheniformis (continuous lines) and B. amyloliquefaciens (dashed lines). Glucose consumption (a), and production of 2,3-butanediol (b), acetoin (c), and ethanol (d).
Applsci 15 07808 g003aApplsci 15 07808 g003b
Table 1. Sugar uptake (%), fermentation products concentration (g/L), biomass OD (600 nm), 2,3-butanediol yield (Y2,3-BDO/sugars, g/g), and 2,3-butanediol productivity (P2,3-BDO, expressed as g/L·h), at the time of maximum 2,3-butanediol production, obtained from B. licheniformis grown in semi-defined media containing different initial glucose concentrations. Data in parentheses refer to sugar uptake at the end of the fermentation process (72 h).
Table 1. Sugar uptake (%), fermentation products concentration (g/L), biomass OD (600 nm), 2,3-butanediol yield (Y2,3-BDO/sugars, g/g), and 2,3-butanediol productivity (P2,3-BDO, expressed as g/L·h), at the time of maximum 2,3-butanediol production, obtained from B. licheniformis grown in semi-defined media containing different initial glucose concentrations. Data in parentheses refer to sugar uptake at the end of the fermentation process (72 h).
Initial Glucose
Conc.
(g/L)		Time
(h)		Sugar Uptake
(%)		2,3-BDO
(g/L)		Ethanol
(g/L)		Acetoin
(g/L)		Biomass OD
(600 nm)		Y2,3-BDO/sugars
(g/g)		P2,3-BDO
(g/L·h)
2012100 (100)2.4 ± 0.01.1 ± 0.05.3 ± 0.06.5 ± 0.10.120.201
4022100 (100)5.2 ± 0.10.7 ± 0.011.7 ± 0.39.2 ± 0.00.130.237
6028100 (100)9.1 ± 0.21.0 ± 0.115.7 ± 0.013.4 ± 0.10.150.323
803285.0 (100)11.2 ± 0.10.8 ± 0.116.8 ± 0.015.0 ± 0.30.170.350
1004694.5 (100)18.0 ± 0.40.9 ± 0.022.5 ± 0.416.2 ± 0.00.200.391
12072100 (100)21.8 ± 0.00.5 ± 0.032.5 ± 0.010.1 ± 0.20.180.303
2,3-butanediol yield (Y2,3-BDO/sugars, g/g): based on the total sugars consumed (in this case, solely glucose).
Table 2. Sugar uptake (%), fermentation products concentration (g/L), biomass OD (600 nm), 2,3-butanediol yield (Y2,3-BDO/sugars, g/g), and 2,3-butanediol productivity (P2,3-BDO, expressed as g/L·h), at the time of maximum 2,3-butanediol production, obtained from B. amyloliquefaciens grown in semi-defined media containing different initial glucose concentrations. Data in parentheses refer to sugar uptake at the end of the fermentation process (144 h).
Table 2. Sugar uptake (%), fermentation products concentration (g/L), biomass OD (600 nm), 2,3-butanediol yield (Y2,3-BDO/sugars, g/g), and 2,3-butanediol productivity (P2,3-BDO, expressed as g/L·h), at the time of maximum 2,3-butanediol production, obtained from B. amyloliquefaciens grown in semi-defined media containing different initial glucose concentrations. Data in parentheses refer to sugar uptake at the end of the fermentation process (144 h).
Initial Glucose Conc.
(g/L)		Time
(h)		Sugar Uptake
(%)		2,3-BDO
(g/L)		Acetoin
(g/L)		Biomass OD
(600 nm)		Y2,3-BDO/sugars
(g/g)		P2,3-BDO
(g/L·h)
2022100 (100)5.8 ± 0.03.3 ± 0.39.8 ± 0.60.290.265
403279.7 (100)11.1 ± 0.33.5 ± 0.313.9 ± 0.30.340.348
605692.8 (100)16.9 ± 0.09.1 ± 0.014.4 ± 0.10.310.302
805662.3 (90.5)15.5 ± 0.07.6 ± 0.112.9 ± 0.80.320.276
1007261.0 (74.2)19.8 ± 0.010.3 ± 0.08.6 ± 0.30.320.275
1205632.2 (39.7)15.0 ± 1.06.8 ± 0.48.0 ± 0.50.390.268
2,3-butanediol yield (Y2,3-BDO/sugars, g/g): based on the total sugars consumed (in this case, solely glucose).
Table 3. Sugar uptake (%), fermentation products concentration (g/L), biomass OD (600 nm), 2,3-butanediol yield (Y2,3-BDO/sugars, g/g) and 2,3-butanediol productivity (P2,3-BDO, expressed as g/L·h), at the time of maximum 2,3-butanediol production, obtained in fermentation assays of carrot discard in SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation) configurations by B. licheniformis and B. amyloliquefaciens. Data in parentheses refer to sugar uptake at the end of the fermentation process (144 h).
Table 3. Sugar uptake (%), fermentation products concentration (g/L), biomass OD (600 nm), 2,3-butanediol yield (Y2,3-BDO/sugars, g/g) and 2,3-butanediol productivity (P2,3-BDO, expressed as g/L·h), at the time of maximum 2,3-butanediol production, obtained in fermentation assays of carrot discard in SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation) configurations by B. licheniformis and B. amyloliquefaciens. Data in parentheses refer to sugar uptake at the end of the fermentation process (144 h).
MicroorganismProcess Conf.Time
(h)		Sugar
Uptake
(%)		2,3-BDO
(g/L)		Ethanol
(g/L)		Acetoin
(g/L)		Biomass OD
(600 nm)		Y2,3-BDO/sugars
(g/g)		P2,3-BDO
(g/L·h)
B. licheniformisSHF46100 (100)10.9 ± 0.80.4 ± 0.324.2 ± 0.418.7 ± 0.00.170.236
SSF2260.2 (51.0)0.9 ± 0.01.5 ± 0.23.7 ± 0.3-0.020.040
B. amyloliquefaciensSHF5681.2 (100)16.7 ± 1.60.2 ± 0.18.0 ± 0.214.6 ± 0.00.330.298
SSF5646.4 (46.4)4.1 ± 0.00.5 ± 0.03.1 ± 0.0-0.140.074
2,3-butanediol yield (Y2,3-BDO/sugars, g/g): based on the total sugars consumed (in this case: glucose, fructose, galactose, and arabinose).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

López-Linares, J.C.; González-Galán, A.M.; Coca, M.; Lucas, S.; García-Cubero, M.T. Harnessing Carrot Discard as a Novel Feedstock for 2,3-Butanediol Bioproduction: A Comparison of Fermentation Strategies and Bacillus Performance. Appl. Sci. 2025, 15, 7808. https://doi.org/10.3390/app15147808

AMA Style

López-Linares JC, González-Galán AM, Coca M, Lucas S, García-Cubero MT. Harnessing Carrot Discard as a Novel Feedstock for 2,3-Butanediol Bioproduction: A Comparison of Fermentation Strategies and Bacillus Performance. Applied Sciences. 2025; 15(14):7808. https://doi.org/10.3390/app15147808

Chicago/Turabian Style

López-Linares, Juan Carlos, Alba Mei González-Galán, Mónica Coca, Susana Lucas, and María Teresa García-Cubero. 2025. "Harnessing Carrot Discard as a Novel Feedstock for 2,3-Butanediol Bioproduction: A Comparison of Fermentation Strategies and Bacillus Performance" Applied Sciences 15, no. 14: 7808. https://doi.org/10.3390/app15147808

APA Style

López-Linares, J. C., González-Galán, A. M., Coca, M., Lucas, S., & García-Cubero, M. T. (2025). Harnessing Carrot Discard as a Novel Feedstock for 2,3-Butanediol Bioproduction: A Comparison of Fermentation Strategies and Bacillus Performance. Applied Sciences, 15(14), 7808. https://doi.org/10.3390/app15147808

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop