# Adaptive Evolution-Assisted Riboflavin Production ofAshbya gossypii from Cane Molasses

Authors: Wenjuan Zhai; Jiantang Cheng; Yuan Li; Jianbin Li; Kai Li; Luolu Wei
Journal: Mathematical Problems in Engineering
 (2022)

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## Abstract

In this study, adaptive evolution was used to improve the adaptation ofAshbya gossypii ATCC 10895 to the substrate (cane molasses) to ensure enhanced substrate utilization and increased riboflavin yield. During the adaptation process, antioxidant activity and riboflavin production ability were evaluated using shake flask fermentation to evaluate the strain response to environmental conditions. After the seventh day of fermentation, results showed that compared with the yield of the parent strain, that of A. gossypii A-24 increased by 97.5%, and the dry cell weight increased by 125%. The A-24 strain was used to further study the cofermentation process of cane molasses and glucose under different ratios. When the ratio of glucose to cane molasses was 1 : 4, the yield of riboflavin was 978 ± 5.37 mg/L, which was 3.7 times that of cane molasses monofermentation.


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## Body

## 1. Introduction

Riboflavin, also known as vitamin B2, is a water-soluble compound that can be synthesized by plants and microorganisms. It is one of the essential micronutrients for the human body, with a wide range of physiological functions. Thus, it has been ranked by the World Health Organization as one of the six main indicators for assessing human growth, development, and nutritional status. At present, riboflavin insufficiency has aroused the concerns of developing and developed countries. It exerts a significant part in cell functions, and it is an indispensable vitamin in life activities. The chemical synthesis for riboflavin has high production costs, serious contamination and safety is questioned, and it is difficult to promote. Thus, microbial fermentation is currently considered the most promising strategy [1–3].At present, microorganisms mainly used for industrial production of riboflavin are mainlyAshbya gossypii and Bacillus subtilis. B. subtilis is a genetically engineered bacterium constructed using genetic engineering, metabolic engineering, and other technical means, and it cannot accumulate riboflavin naturally. Industrial bacteria constructed based on new breeding techniques, such as DNA recombination and genome rearrangement, require using antibiotic genes during the construction process. The use of these resistance genes is very unfavorable for food-grade or feed-grade riboflavin products and is likely to cause proliferation of antibiotics, especially in the case of incomplete poststerilization processes [4–6]. In 2001, The European Food Safety Authority states that neither GMM nor its recombinant DNA must be present in the final product that is placed on the EU market as non-GMM food or feed additive. A. gossypii is a natural riboflavin-producing strain with simple nutritional requirements. It is nonpathogenic and meets food safety requirements, and it can be used as the main riboflavin-producing strain [7].Vegetable oil and glucose are currently the main raw materials for the production of riboflavin. Due to the competition in riboflavin and nutritional security, especially in developing countries, considerable debate about its sustainability has been present. Therefore, seeking novel raw materials to produce riboflavin without affecting the agricultural sector and food security has become a key research issue. In this context, cane molasses has received increased attention for it is a low-cost food crop with no competing riboflavin carbon sources [8–10].Sugarcane molasses is a byproduct of approximately 3 million tons of annual output of sugar factories in my country. It contains approximately 50% (w/w) of sugars (mainly sucrose, fructose, and glucose), suspended colloids, and a minor amount of nitrogen compounds, minerals, and metal ions. Compared with other inexpensive substrates, cane molasses is abundant in nutrients. In addition, cane molasses that is produced in large quantities may cause environmental pollution if not processed in time. Therefore, it is used to produce various biological products, such as lactic acid, butyric acid, and propionic acid. Notably, cane molasses is quite inhibitory to microorganisms due to the presence of toxic elements, such as 5-hydroxymethyl furfural and excess metal ions. These substances complicate the deployment of industrial strains by using cane molasses as the raw material [11–13].Dual-substrate mixed fermentation is essential to improve the utilization of the substrate and the concentration of the target product. Strains widely used in riboflavin fermentation are easily inhibited by substances in cane molasses. Therefore, enhancing strain tolerance is necessary for the production of riboflavin from untreated cane molasses [14]. Adaptive evolution is the cultivation of microorganisms under specific environmental conditions in specific laboratories to promote the adaptive natural evolution of microorganisms and finally obtain target strains with beneficial mutations through screening. It is an effective method to develop microorganisms with industrial ideal phenotypes. In the previous study, the adaptation of Lactobacillus paracasei NRRL B-4564 to the substrate was improved, the utilization of the substrate was strengthened, and the production of L(+) lactic acid in the dead residue of molasses-rich potato was improved [15, 16].In this study, the method of batch adaptation evolution was used to improve the tolerance ofA. gossypii strain. Cane molasses was added in increasing concentrations during the culture process. In the 240 g/L cane molasses medium, the effect of adaptation conditions on the growth and fermentation performance of the strain was studied. In addition, to further improve the utilization rate of cane molasses and the yield of riboflavin, the strain of cane molasses/glucose-mixed fermentation riboflavin was studied. On this basis, the effects of cofermentation on riboflavin, cell dry weight, substrate consumption, and main metabolic pathways were studied [17–20].


## 2. Materials and Methods

### 2.1. Strains and Media

A. gossypii ATCC 10895 was purchased from Mingzhou Corporation Ningbo. The strain preserved at −80°C was activated and cultured in the AFM medium. AFM (pH 6.8) containing 10 g/L yeast extract, 10 g/L tryptone, 1 g/L inositol, and 20 g/L glucose was used as the seed medium for riboflavin production. Seed culture was carried out at 28°C on a rotary shaker (BSD-YX2400, Shanghai, China) at 220 rpm for 48 h. For riboflavin production in flask cultures, 5 ml of seed culture was inoculated into 500 ml Erlenmeyer flasks containing 50 ml of production medium. Cultivation was carried out using a 500 ml flask (working volume of 50 ml) with an agitation rate of 220 rpm at 28°C.


### 2.2. Adaptation ofA. gossypii to Cane Molasses

A. gossypii was activated and placed in the AFM medium. After the strain grew to the late exponential stage, it was transferred to the 150 g/L selective medium. The operation was repeated three times. The molasses concentration of the selective medium was gradually increased by 30 g/L unit until the strain no longer grew to enhance its adaptability to molasses. Adaption methods performed for the preparation of adapted A. gossypii were tested, as shown in Figure 1. During the evolution process, a certain amount of bacterial liquid was taken and stored in the refrigerator at −80°C every time. The evolution of the strain was investigated by measuring its growth curve.Figure 1
Schematic of the adaptation process.


### 2.3. Determination of Antioxidant Activity of Parental and Adaptive Strains

#### 2.3.1. Preparation of Intact Cells, Intracellular Cell-Free Extracts, and Supernatant

For the evaluation of antioxidant activity, parents and four adapted strains were grown in AFM broth supplemented with 24% (w/v) of cane molasses. After 4 days of incubation at 28°C under microaerophilic static conditions, the cells were harvested by centrifugation (10000× g, 10 min, centrifuge: Boxun, Shanghai, China), washed triple times in deionized water, and resuspended in deionized water to make intact cells. The intracellular cell-free extracts were prepared using the method of Lin and Yen. Cell disruption was performed using an ultrasonic homogenizer (SCIENTZ, Ningbo, China). Cell debris was removed by centrifugation (10000× g, 10 min, 4°C), and the resulting supernatant was used for antioxidant activity assay.


#### 2.3.2. DPPH Free-Radical Scavenging Activity

In accordance with the method of Li with some modifications, the 2,2-diphenyl-1-picrylhydrazyl DPPH free-radical scavenging ability of the parent and adapted strains were determined. In brief, 1 ml of sample (intact cells, intracellular cell-free extracts, and supernatant) was added to 1 ml of freshly prepared DPPH solution (0.15 mM in ethanol). The mixture was forced and incubated at room temperature in the dark for 30 min. The blanks contained the sample and ethanol, while the controls included ethanol and DPPH solution. After centrifugation (12,000× g, 10 min, 4°C), the absorbance of DPPH radical at 517 nm was determined. The scavenging ability was defined as follows:(1)Scavenging activity%=1−Asample−Ablank/Acontrol×100.


### 2.4. Riboflavin Production from Cane Molasses and Glucose

The untreated cane molasses (diluted to the required sugar concentration) was used as the substrate for riboflavin production. The effects of the initial sugar concentration of cane molasses (180, 200, 220, 240, and 260 g/L) were also evaluated in the Erlenmeyer flask. All the experiments were performed at least three times.


### 2.5. Riboflavin Production by Cofermentation of Cane Molasses and Glucose

Cofermentation assays were prepared as described above for the riboflavin production assays, using different cane molasses and glucose ratios (5 : 0, 4 : 1, 3 : 2, 2 : 3, 1 : 4, and 0 : 5) with a final total sugar concentration of 40 g/L. The medium was autoclaved at 115°C for 30 min. The inoculation amount was 10% (v/v). The experiment was carried out in a 500 ml Erlenmeyer flask containing 50 ml medium, and these cultures were grown for 7 days at 28°C on a rotary at 220 rpm. Riboflavin production was measured every other day.


### 2.6. Analytical Methods

The amount of riboflavin was determined in accordance with a previous protocol (Tajima et al., 2009). In brief, 0.8 mL of the culture broth was thoroughly mixed with 0.2 mL of 1 N NaOH. Then, 0.4 mL aliquot of the resulting solution was neutralized with 1 mL of 0.1 M potassium phosphate buffer (pH 6.0), and the absorbance of the solution at a wavelength of 444 nm was measured.Sugars (sucrose, fructose, and glucose) and organic acids (citric acid, acetic acid, D-gluconic acid, succinic acid, and pyruvic acid) were determined by high-performance liquid chromatography (HPLC; Agilent, Santa Clara, USA) with Aminex HPX-87H (BIO-RAD, Beijing, China) at 50°C and a refractive index detector (Agilent, Santa Clara, USA) at 50°C. 5 mM of H2SO4 solution at 0.6 mL/min was used as the mobile phase.


## 2.1. Strains and Media

A. gossypii ATCC 10895 was purchased from Mingzhou Corporation Ningbo. The strain preserved at −80°C was activated and cultured in the AFM medium. AFM (pH 6.8) containing 10 g/L yeast extract, 10 g/L tryptone, 1 g/L inositol, and 20 g/L glucose was used as the seed medium for riboflavin production. Seed culture was carried out at 28°C on a rotary shaker (BSD-YX2400, Shanghai, China) at 220 rpm for 48 h. For riboflavin production in flask cultures, 5 ml of seed culture was inoculated into 500 ml Erlenmeyer flasks containing 50 ml of production medium. Cultivation was carried out using a 500 ml flask (working volume of 50 ml) with an agitation rate of 220 rpm at 28°C.


## 2.2. Adaptation ofA. gossypii to Cane Molasses

A. gossypii was activated and placed in the AFM medium. After the strain grew to the late exponential stage, it was transferred to the 150 g/L selective medium. The operation was repeated three times. The molasses concentration of the selective medium was gradually increased by 30 g/L unit until the strain no longer grew to enhance its adaptability to molasses. Adaption methods performed for the preparation of adapted A. gossypii were tested, as shown in Figure 1. During the evolution process, a certain amount of bacterial liquid was taken and stored in the refrigerator at −80°C every time. The evolution of the strain was investigated by measuring its growth curve.Figure 1
Schematic of the adaptation process.


## 2.3. Determination of Antioxidant Activity of Parental and Adaptive Strains

### 2.3.1. Preparation of Intact Cells, Intracellular Cell-Free Extracts, and Supernatant

For the evaluation of antioxidant activity, parents and four adapted strains were grown in AFM broth supplemented with 24% (w/v) of cane molasses. After 4 days of incubation at 28°C under microaerophilic static conditions, the cells were harvested by centrifugation (10000× g, 10 min, centrifuge: Boxun, Shanghai, China), washed triple times in deionized water, and resuspended in deionized water to make intact cells. The intracellular cell-free extracts were prepared using the method of Lin and Yen. Cell disruption was performed using an ultrasonic homogenizer (SCIENTZ, Ningbo, China). Cell debris was removed by centrifugation (10000× g, 10 min, 4°C), and the resulting supernatant was used for antioxidant activity assay.


### 2.3.2. DPPH Free-Radical Scavenging Activity

In accordance with the method of Li with some modifications, the 2,2-diphenyl-1-picrylhydrazyl DPPH free-radical scavenging ability of the parent and adapted strains were determined. In brief, 1 ml of sample (intact cells, intracellular cell-free extracts, and supernatant) was added to 1 ml of freshly prepared DPPH solution (0.15 mM in ethanol). The mixture was forced and incubated at room temperature in the dark for 30 min. The blanks contained the sample and ethanol, while the controls included ethanol and DPPH solution. After centrifugation (12,000× g, 10 min, 4°C), the absorbance of DPPH radical at 517 nm was determined. The scavenging ability was defined as follows:(1)Scavenging activity%=1−Asample−Ablank/Acontrol×100.


## 2.3.1. Preparation of Intact Cells, Intracellular Cell-Free Extracts, and Supernatant

For the evaluation of antioxidant activity, parents and four adapted strains were grown in AFM broth supplemented with 24% (w/v) of cane molasses. After 4 days of incubation at 28°C under microaerophilic static conditions, the cells were harvested by centrifugation (10000× g, 10 min, centrifuge: Boxun, Shanghai, China), washed triple times in deionized water, and resuspended in deionized water to make intact cells. The intracellular cell-free extracts were prepared using the method of Lin and Yen. Cell disruption was performed using an ultrasonic homogenizer (SCIENTZ, Ningbo, China). Cell debris was removed by centrifugation (10000× g, 10 min, 4°C), and the resulting supernatant was used for antioxidant activity assay.


## 2.3.2. DPPH Free-Radical Scavenging Activity

In accordance with the method of Li with some modifications, the 2,2-diphenyl-1-picrylhydrazyl DPPH free-radical scavenging ability of the parent and adapted strains were determined. In brief, 1 ml of sample (intact cells, intracellular cell-free extracts, and supernatant) was added to 1 ml of freshly prepared DPPH solution (0.15 mM in ethanol). The mixture was forced and incubated at room temperature in the dark for 30 min. The blanks contained the sample and ethanol, while the controls included ethanol and DPPH solution. After centrifugation (12,000× g, 10 min, 4°C), the absorbance of DPPH radical at 517 nm was determined. The scavenging ability was defined as follows:(1)Scavenging activity%=1−Asample−Ablank/Acontrol×100.


## 2.4. Riboflavin Production from Cane Molasses and Glucose

The untreated cane molasses (diluted to the required sugar concentration) was used as the substrate for riboflavin production. The effects of the initial sugar concentration of cane molasses (180, 200, 220, 240, and 260 g/L) were also evaluated in the Erlenmeyer flask. All the experiments were performed at least three times.


## 2.5. Riboflavin Production by Cofermentation of Cane Molasses and Glucose

Cofermentation assays were prepared as described above for the riboflavin production assays, using different cane molasses and glucose ratios (5 : 0, 4 : 1, 3 : 2, 2 : 3, 1 : 4, and 0 : 5) with a final total sugar concentration of 40 g/L. The medium was autoclaved at 115°C for 30 min. The inoculation amount was 10% (v/v). The experiment was carried out in a 500 ml Erlenmeyer flask containing 50 ml medium, and these cultures were grown for 7 days at 28°C on a rotary at 220 rpm. Riboflavin production was measured every other day.


## 2.6. Analytical Methods

The amount of riboflavin was determined in accordance with a previous protocol (Tajima et al., 2009). In brief, 0.8 mL of the culture broth was thoroughly mixed with 0.2 mL of 1 N NaOH. Then, 0.4 mL aliquot of the resulting solution was neutralized with 1 mL of 0.1 M potassium phosphate buffer (pH 6.0), and the absorbance of the solution at a wavelength of 444 nm was measured.Sugars (sucrose, fructose, and glucose) and organic acids (citric acid, acetic acid, D-gluconic acid, succinic acid, and pyruvic acid) were determined by high-performance liquid chromatography (HPLC; Agilent, Santa Clara, USA) with Aminex HPX-87H (BIO-RAD, Beijing, China) at 50°C and a refractive index detector (Agilent, Santa Clara, USA) at 50°C. 5 mM of H2SO4 solution at 0.6 mL/min was used as the mobile phase.


## 3. Results and Discussion

### 3.1. Adaptive Evolution ofA. gossypii

The evolutionary adaption of microorganisms to cane molasses is an effective method to improve inhibitor tolerance and fermentation performance. In this study, a stepwise adaptation strategy by continuously transferring the strains to the adaptation medium with increased cane molasses concentration was studied. Four different adaptation methods were tested, and finally, the adapted strains were inoculated into the fermentation medium with a cane molasses concentration of 240 g/L.As shown in Figure2, the parent strain had an obvious lag period. After 7 days of fermentation, only 149 mg/L of riboflavin could be obtained (Figure 2(a)). The ability of the adapted strain to produce riboflavin was significantly better than that of the parent strain (Figure 2(a)), and the riboflavin production of the strain gradually increased with time. During the 7-day fermentation process of A-24, the best riboflavin yield reached 298 mg/L. The riboflavin production performance of the strain and its antioxidant capacity showed good consistency, indicating that the enhanced protection of the strain against harmful substances in the culture medium and its effective adaptation to waste substrates are the reasons for the increased production of riboflavin. Figures 2(b) and 2(c) show the dry cell weight and total sugar content of the parent strain and the adapted strain. Similar to the yield of riboflavin, the adapted strain’s dry weight and substrate utilization rate are better than those of the parent strain. Figure 2(d) shows the growth rates of different adapted strains compared to those of the parent strains in terms of riboflavin production, dry cell weight, and substrate utilization. The results showed that compared with the riboflavin production of the parent strain, that of the adaptive strain (A-15, A-18, A-21, and A-24) increased by 19.6%, 30.3%, 51.4%, and 100%, respectively, and the dry cell weight increased by 5.5%, 17.3%, 19.4%, and 125%, respectively. Among the four selected strains, the growth performance of strain A-24 was significantly improved due to the previous effective cell growth regulation.Figure 2
Riboflavin fermentation of cane molasses by parent and adaptive strains. Time course of riboflavin produced (a) dry cell weight (b) total sugar concentration (c) and adapted to the growth rate of riboflavin and stem weight of strain relative to the parent (d).
(a)(b)(c)(d)


### 3.2. Effect of Adaptability on the Antioxidant Activity

Strains with extensive substrate utilization capabilities and resistance to undesirable compounds or stress conditions are required to ensure effective production of riboflavin on complex substrates. The antioxidant activity of the parent and adapted strains selected in the course of adaptation was further evaluated to illustrate the response of cells to environmental condition. The antioxidant activities of the parent strain upon exposure to different environments (AFM broth supplemented with 24% cane molasses) and adapted strains (A-15, A-18, A-21, and A-24) are presented in Figure3. The results showed that the riboflavin production capacity is related to its antioxidant capacity.Figure 3
DPPH free-radical scavenging ability of parent and adapted strains. Bars labeled with different letters indicate significant difference (Tukey’s test,p<0.05).The elimination of DPPH free radicals is due to the hydrogen supply capacity of antioxidants, and it is the most commonly used method to evaluate the antioxidant capacity. In general, strains exposed to high molasses concentration cause high antioxidant activity. As shown in Figure3, the DPPH free-radical scavenging rate of all strains is in the following order: supernatant > intact cells > cell extract. The antioxidation of intact cell and supernatant was higher than that of intracellular cell-free extracts, possibly due to intact bacteria. The antioxidant enzyme system is contained in the cell itself, and the free-radical scavenging ability of the fermentation supernatant comes from substances with antioxidant activity, such as riboflavin, extracellular polysaccharides, and antioxidant peptides. Polysaccharides can be used as electron donors to combine free-radical ions neutralized with DPPH free radicals, and the chain reaction must be terminated while removing free radicals. The low DPPH free-radical scavenging activity of intracellular cell-free extracts may be related to the loss of activity of intracellular antioxidant enzymes during cell lysis.The DPPH free-radical scavenging abilities of the parent strain were 30.5% (intracellular cell-free extracts), 33.7% (intact cells), and 57.5% (supernatant). The adaptedA. gossypii (A-15, A-22, and A-24) showed even higher DPPH radical scavenging ability in the range of 67.1%–85.9% for intracellular cell-free extracts, 48.5%–61.7% for intact cells, and 72%–94% for supernatant (Figure 3). The DPPH free-radical scavenging ability of A-24 was significantly higher than that of the parent strain. The increase in antioxidant capacity of the tested strains subjected to cane molasses indicated that the strain exposure to molasses induced the synthesis of antioxidant metabolites and enzymes involved in DPPH free-radical scavenging. In addition, heavy metals, furan aldehydes, and different phenolic compounds induce the accumulation of ROS (hydrogen peroxide, superoxide anion, and hydroxyl radicals) in many microorganisms. Therefore, the improvement of antioxidant defense capacity in A. gossypii in this study could be considered a response to various pro-oxidant compounds present in cane molasses, such as furan aldehydes, phenolic compounds, volatile compounds, and metal ions. On the basis of this study, the adaptive strain A-24 was chosen for the following fermentation evaluation.


### 3.3. Optimization of Cane Molasses

The culture medium is the basic guarantee for the growth of microorganisms. It affects the growth of bacteria, and it is closely related to the formation of metabolites. The carbon source is the basic element for the growth of bacteria, the raw material for the production of various metabolites, and the energy source for microorganisms. Therefore, selecting an appropriate carbon source concentration for the growth of the bacteria and the synthesis of the target product is very important.The influence of cane molasses concentration (by diluting cane molasses with water from 180 g/L to 260 g/L) on riboflavin production was studied in shake flasks to evaluate the effect of untreated cane molasses on A-24 fermentation. As shown in Figures4(a)–4(c), as the concentration of cane molasses increased from 180 g/L to 220 g/L, the concentration of riboflavin increased from 293.78 ± 5.42 mg/L to 326.44 ± 4.79 mg/L. Further increasing the sugar concentration to 260 g/L resulted in a decrease in the concentration of riboflavin (219.92 ± 0.09 mg/L), which could be attributed to the high concentration of substrates in untreated sugarcane molasses.Figure 4
The influence of cane molasses concentration.
(a)(b)(c)


### 3.4. Coordination Optimization

Cofermentation is an efficient fermentation strategy that increases riboflavin yield by providing multiple substrates. Therefore, riboflavin production test was conducted under different ratios of cane molasses and glucose (4 : 1, 3 : 2, 2 : 3, and 1 : 4) in comparison with the individual substrates performed (cane molasses : glucose ratio of 5 : 0 and 0 : 5). Based on the past experience, the total sugar concentration was 40 g/L. This experiment was carried out under the condition of changing the ratio of carbon source in the medium, as shown in Table1.Table 1
Basic parameters of each experimental group.
Ratio of sugarcane molasses to glucoseCane molasses (g/L)Glucose (g/L)5 : 016604 : 113283 : 299162 : 366241 : 433320 : 5040As shown in Figure5(a), the highest yield of riboflavin was obtained in the single-substrate glucose fermentation experiment, while the single-substrate sugarcane molasses obtained the lowest yield. The yield of the cofermentation mixture was much higher than that of the cane molasses control (5 : 0). The higher yield of glucose in single fermentation than in cofermentation is due to the inhibition of carbon catabolism. As the proportion of glucose in the cofermentation increases, the output of the cofermentation mixture increases. Among them, the yields of cane molasses/glucose (1 : 4 and 2 : 3) were significantly higher than those of cane molasses at 400 and 1020 mg/L, respectively. The fermentation yield increased with the increase in glucose ratio, indicating that under the cofermentation conditions, the increase in fermentation yield may be mainly related to the increase in the degree of glucose fermentation. A notable detail is that in single glucose fermentation, in terms of cell dry weight, total sugar was consumed in large quantities for cell metabolism because sugarcane molasses is more conducive to cell growth than glucose, resulting in a downward trend in cell dry weight after 72 h (Figure 5(b)), 1 : 4 and 2 : 3. After 72 h of fermentation, the total sugar consumption became stable, the cell metabolism was lower than that of single glucose fermentation, and the cell dry weight tended to be stable. However, for single cane molasses fermentation, the total sugar consumption was slow before 72 h, and cell metabolism was slow, resulting in its yield and cell dry weight being much lower than those in cofermentation (Figure 5(c)). On the basis of these results, a hypothesis could be put forward. For A. gossypii, glucose is more conducive to cell metabolism than sugarcane molasses, and it increases the yield of the target product.Figure 5
Test curves of sugarcane molasses and glucose in different proportions.
(a)(b)(c)


### 3.5. Effect of Cofermentation on Fermentation By-Products

The metabolic pathways involved in riboflavin synthesis include glycolysis, pentose phosphate pathway, and purine synthesis and riboflavin synthesis pathway. Studying the pathways of sugar metabolism is essential for the regulation of riboflavin anabolic metabolism. The key nodes of sugar metabolism are all related to organic acid metabolism. The use of HPLC to determine the accumulation of organic acids could indirectly infer the metabolic regulation mechanism of the bacteria during different fermentation periods. Gluconic acid is the product of phosphate glucose dehydrogenase, a key enzyme of the pentose phosphate pathway. Pyruvate is the end product of the glycolysis pathway, and it could be converted into acetyl-CoA. Citric acid and succinic acid are intermediates of the citric acid cycle.The normal growth of bacteria mainly depends on the EMP pathway and the TCA cycle for maintenance, and the synthesis of riboflavin requires the HMP pathway to provide the precursors 5-phosphoribulose and 5-phosphoribose (for de-novo synthesis of GTP). In this study, no gluconic acid was detected in all cofermentation processes (Figures6(a)–6(f)). Figures 6(c)–6(f) show that when the ratio of cane molasses to cofermentation was 2 : 3, the production time of citric acid and pyruvic acid was delayed (appeared after 2 days), and as the concentration of cane molasses in cofermentation increased, the time of organic acid appearance was gradually delayed. When the proportion of cane molasses in cofermentation was greater than 80%, succinic acid could not be detected in the whole fermentation process. During dual-substrate fermentation, as the ratio of cane molasses content increased, the carbon metabolism flow in the EMP pathway and TCA decreased, and the substrate utilization rate gradually decreased. When the glucose content in the cofermentation ratio was increased, the TCA and EMP pathways showed higher carbon fluxes than cane molasses, the production of riboflavin increased, and the bacterial cells demonstrated a high utilization rate of mixed substrates. The distribution of organic acids exhibited that adding glucose increased not only the carbon metabolic flow of the TCA and EMP pathways but also the metabolic flow of the riboflavin synthesis pathway.Figure 6
Effect of cofermentation on fermentation by-products.
(a)(b)(c)(d)(e)(f)


### 3.6. Morphological Changes in Bacteria during Cofermentation

When cultured in glucose monofermentation for 24 h, the elongated hyphae appeared as a septum, dividing the hyphae into several small compartments (Figure7(a)). However, the grown hyphae began to consume nutrients and accumulate within themselves, thus thickening the hyphae and increasing the concentration of riboflavin inside the hyphae (Figure 7(b)). The produced riboflavin leaked out of the bacteria, and the hyphae became empty or formed intracellular crystals (riboflavin has low solubility in water) (Figures 7(c) and 7(d)). After 96 h of cultivation, riboflavin agglomerated in large quantities outside the hyphae, and a large number of riboflavin crystals appeared in the fermentation broth (Figure 7(e)). After 120 h of fermentation, sporangia appeared in the bacterial aging process (Figure 7(f)). The cofermentation mixture (cane molasses : glucose was 1 : 4) showed riboflavin crystals 24 h later than the whole glucose, while the other mixtures (2 : 3, 3 : 2, and 4 : 1) only showed intracellular riboflavin crystals at 120 h.Figure 7
Micrograph of morphological changes of bacteria during cofermentation.When cultured in cane molasses monofermentation (as shown in Figures7(g) and 7(h)), although no glucose was added for culture, the culture conditions were the same, the hypha became thinner, and the accumulation of intracellular riboflavin was lower than when cultured with whole glucose. No intracellular crystals of inner nuclear flavor were seen. When cultured for 48 h, the hyphae appeared as a septum 24 h later than the full glucose medium. At 96 h, the cell debris appeared as extracellular riboflavin (Figure 7(h)), which was absent in the full glucose medium and 1 : 4.


## 3.1. Adaptive Evolution ofA. gossypii

The evolutionary adaption of microorganisms to cane molasses is an effective method to improve inhibitor tolerance and fermentation performance. In this study, a stepwise adaptation strategy by continuously transferring the strains to the adaptation medium with increased cane molasses concentration was studied. Four different adaptation methods were tested, and finally, the adapted strains were inoculated into the fermentation medium with a cane molasses concentration of 240 g/L.As shown in Figure2, the parent strain had an obvious lag period. After 7 days of fermentation, only 149 mg/L of riboflavin could be obtained (Figure 2(a)). The ability of the adapted strain to produce riboflavin was significantly better than that of the parent strain (Figure 2(a)), and the riboflavin production of the strain gradually increased with time. During the 7-day fermentation process of A-24, the best riboflavin yield reached 298 mg/L. The riboflavin production performance of the strain and its antioxidant capacity showed good consistency, indicating that the enhanced protection of the strain against harmful substances in the culture medium and its effective adaptation to waste substrates are the reasons for the increased production of riboflavin. Figures 2(b) and 2(c) show the dry cell weight and total sugar content of the parent strain and the adapted strain. Similar to the yield of riboflavin, the adapted strain’s dry weight and substrate utilization rate are better than those of the parent strain. Figure 2(d) shows the growth rates of different adapted strains compared to those of the parent strains in terms of riboflavin production, dry cell weight, and substrate utilization. The results showed that compared with the riboflavin production of the parent strain, that of the adaptive strain (A-15, A-18, A-21, and A-24) increased by 19.6%, 30.3%, 51.4%, and 100%, respectively, and the dry cell weight increased by 5.5%, 17.3%, 19.4%, and 125%, respectively. Among the four selected strains, the growth performance of strain A-24 was significantly improved due to the previous effective cell growth regulation.Figure 2
Riboflavin fermentation of cane molasses by parent and adaptive strains. Time course of riboflavin produced (a) dry cell weight (b) total sugar concentration (c) and adapted to the growth rate of riboflavin and stem weight of strain relative to the parent (d).
(a)(b)(c)(d)


## 3.2. Effect of Adaptability on the Antioxidant Activity

Strains with extensive substrate utilization capabilities and resistance to undesirable compounds or stress conditions are required to ensure effective production of riboflavin on complex substrates. The antioxidant activity of the parent and adapted strains selected in the course of adaptation was further evaluated to illustrate the response of cells to environmental condition. The antioxidant activities of the parent strain upon exposure to different environments (AFM broth supplemented with 24% cane molasses) and adapted strains (A-15, A-18, A-21, and A-24) are presented in Figure3. The results showed that the riboflavin production capacity is related to its antioxidant capacity.Figure 3
DPPH free-radical scavenging ability of parent and adapted strains. Bars labeled with different letters indicate significant difference (Tukey’s test,p<0.05).The elimination of DPPH free radicals is due to the hydrogen supply capacity of antioxidants, and it is the most commonly used method to evaluate the antioxidant capacity. In general, strains exposed to high molasses concentration cause high antioxidant activity. As shown in Figure3, the DPPH free-radical scavenging rate of all strains is in the following order: supernatant > intact cells > cell extract. The antioxidation of intact cell and supernatant was higher than that of intracellular cell-free extracts, possibly due to intact bacteria. The antioxidant enzyme system is contained in the cell itself, and the free-radical scavenging ability of the fermentation supernatant comes from substances with antioxidant activity, such as riboflavin, extracellular polysaccharides, and antioxidant peptides. Polysaccharides can be used as electron donors to combine free-radical ions neutralized with DPPH free radicals, and the chain reaction must be terminated while removing free radicals. The low DPPH free-radical scavenging activity of intracellular cell-free extracts may be related to the loss of activity of intracellular antioxidant enzymes during cell lysis.The DPPH free-radical scavenging abilities of the parent strain were 30.5% (intracellular cell-free extracts), 33.7% (intact cells), and 57.5% (supernatant). The adaptedA. gossypii (A-15, A-22, and A-24) showed even higher DPPH radical scavenging ability in the range of 67.1%–85.9% for intracellular cell-free extracts, 48.5%–61.7% for intact cells, and 72%–94% for supernatant (Figure 3). The DPPH free-radical scavenging ability of A-24 was significantly higher than that of the parent strain. The increase in antioxidant capacity of the tested strains subjected to cane molasses indicated that the strain exposure to molasses induced the synthesis of antioxidant metabolites and enzymes involved in DPPH free-radical scavenging. In addition, heavy metals, furan aldehydes, and different phenolic compounds induce the accumulation of ROS (hydrogen peroxide, superoxide anion, and hydroxyl radicals) in many microorganisms. Therefore, the improvement of antioxidant defense capacity in A. gossypii in this study could be considered a response to various pro-oxidant compounds present in cane molasses, such as furan aldehydes, phenolic compounds, volatile compounds, and metal ions. On the basis of this study, the adaptive strain A-24 was chosen for the following fermentation evaluation.


## 3.3. Optimization of Cane Molasses

The culture medium is the basic guarantee for the growth of microorganisms. It affects the growth of bacteria, and it is closely related to the formation of metabolites. The carbon source is the basic element for the growth of bacteria, the raw material for the production of various metabolites, and the energy source for microorganisms. Therefore, selecting an appropriate carbon source concentration for the growth of the bacteria and the synthesis of the target product is very important.The influence of cane molasses concentration (by diluting cane molasses with water from 180 g/L to 260 g/L) on riboflavin production was studied in shake flasks to evaluate the effect of untreated cane molasses on A-24 fermentation. As shown in Figures4(a)–4(c), as the concentration of cane molasses increased from 180 g/L to 220 g/L, the concentration of riboflavin increased from 293.78 ± 5.42 mg/L to 326.44 ± 4.79 mg/L. Further increasing the sugar concentration to 260 g/L resulted in a decrease in the concentration of riboflavin (219.92 ± 0.09 mg/L), which could be attributed to the high concentration of substrates in untreated sugarcane molasses.Figure 4
The influence of cane molasses concentration.
(a)(b)(c)


## 3.4. Coordination Optimization

Cofermentation is an efficient fermentation strategy that increases riboflavin yield by providing multiple substrates. Therefore, riboflavin production test was conducted under different ratios of cane molasses and glucose (4 : 1, 3 : 2, 2 : 3, and 1 : 4) in comparison with the individual substrates performed (cane molasses : glucose ratio of 5 : 0 and 0 : 5). Based on the past experience, the total sugar concentration was 40 g/L. This experiment was carried out under the condition of changing the ratio of carbon source in the medium, as shown in Table1.Table 1
Basic parameters of each experimental group.
Ratio of sugarcane molasses to glucoseCane molasses (g/L)Glucose (g/L)5 : 016604 : 113283 : 299162 : 366241 : 433320 : 5040As shown in Figure5(a), the highest yield of riboflavin was obtained in the single-substrate glucose fermentation experiment, while the single-substrate sugarcane molasses obtained the lowest yield. The yield of the cofermentation mixture was much higher than that of the cane molasses control (5 : 0). The higher yield of glucose in single fermentation than in cofermentation is due to the inhibition of carbon catabolism. As the proportion of glucose in the cofermentation increases, the output of the cofermentation mixture increases. Among them, the yields of cane molasses/glucose (1 : 4 and 2 : 3) were significantly higher than those of cane molasses at 400 and 1020 mg/L, respectively. The fermentation yield increased with the increase in glucose ratio, indicating that under the cofermentation conditions, the increase in fermentation yield may be mainly related to the increase in the degree of glucose fermentation. A notable detail is that in single glucose fermentation, in terms of cell dry weight, total sugar was consumed in large quantities for cell metabolism because sugarcane molasses is more conducive to cell growth than glucose, resulting in a downward trend in cell dry weight after 72 h (Figure 5(b)), 1 : 4 and 2 : 3. After 72 h of fermentation, the total sugar consumption became stable, the cell metabolism was lower than that of single glucose fermentation, and the cell dry weight tended to be stable. However, for single cane molasses fermentation, the total sugar consumption was slow before 72 h, and cell metabolism was slow, resulting in its yield and cell dry weight being much lower than those in cofermentation (Figure 5(c)). On the basis of these results, a hypothesis could be put forward. For A. gossypii, glucose is more conducive to cell metabolism than sugarcane molasses, and it increases the yield of the target product.Figure 5
Test curves of sugarcane molasses and glucose in different proportions.
(a)(b)(c)


## 3.5. Effect of Cofermentation on Fermentation By-Products

The metabolic pathways involved in riboflavin synthesis include glycolysis, pentose phosphate pathway, and purine synthesis and riboflavin synthesis pathway. Studying the pathways of sugar metabolism is essential for the regulation of riboflavin anabolic metabolism. The key nodes of sugar metabolism are all related to organic acid metabolism. The use of HPLC to determine the accumulation of organic acids could indirectly infer the metabolic regulation mechanism of the bacteria during different fermentation periods. Gluconic acid is the product of phosphate glucose dehydrogenase, a key enzyme of the pentose phosphate pathway. Pyruvate is the end product of the glycolysis pathway, and it could be converted into acetyl-CoA. Citric acid and succinic acid are intermediates of the citric acid cycle.The normal growth of bacteria mainly depends on the EMP pathway and the TCA cycle for maintenance, and the synthesis of riboflavin requires the HMP pathway to provide the precursors 5-phosphoribulose and 5-phosphoribose (for de-novo synthesis of GTP). In this study, no gluconic acid was detected in all cofermentation processes (Figures6(a)–6(f)). Figures 6(c)–6(f) show that when the ratio of cane molasses to cofermentation was 2 : 3, the production time of citric acid and pyruvic acid was delayed (appeared after 2 days), and as the concentration of cane molasses in cofermentation increased, the time of organic acid appearance was gradually delayed. When the proportion of cane molasses in cofermentation was greater than 80%, succinic acid could not be detected in the whole fermentation process. During dual-substrate fermentation, as the ratio of cane molasses content increased, the carbon metabolism flow in the EMP pathway and TCA decreased, and the substrate utilization rate gradually decreased. When the glucose content in the cofermentation ratio was increased, the TCA and EMP pathways showed higher carbon fluxes than cane molasses, the production of riboflavin increased, and the bacterial cells demonstrated a high utilization rate of mixed substrates. The distribution of organic acids exhibited that adding glucose increased not only the carbon metabolic flow of the TCA and EMP pathways but also the metabolic flow of the riboflavin synthesis pathway.Figure 6
Effect of cofermentation on fermentation by-products.
(a)(b)(c)(d)(e)(f)


## 3.6. Morphological Changes in Bacteria during Cofermentation

When cultured in glucose monofermentation for 24 h, the elongated hyphae appeared as a septum, dividing the hyphae into several small compartments (Figure7(a)). However, the grown hyphae began to consume nutrients and accumulate within themselves, thus thickening the hyphae and increasing the concentration of riboflavin inside the hyphae (Figure 7(b)). The produced riboflavin leaked out of the bacteria, and the hyphae became empty or formed intracellular crystals (riboflavin has low solubility in water) (Figures 7(c) and 7(d)). After 96 h of cultivation, riboflavin agglomerated in large quantities outside the hyphae, and a large number of riboflavin crystals appeared in the fermentation broth (Figure 7(e)). After 120 h of fermentation, sporangia appeared in the bacterial aging process (Figure 7(f)). The cofermentation mixture (cane molasses : glucose was 1 : 4) showed riboflavin crystals 24 h later than the whole glucose, while the other mixtures (2 : 3, 3 : 2, and 4 : 1) only showed intracellular riboflavin crystals at 120 h.Figure 7
Micrograph of morphological changes of bacteria during cofermentation.When cultured in cane molasses monofermentation (as shown in Figures7(g) and 7(h)), although no glucose was added for culture, the culture conditions were the same, the hypha became thinner, and the accumulation of intracellular riboflavin was lower than when cultured with whole glucose. No intracellular crystals of inner nuclear flavor were seen. When cultured for 48 h, the hyphae appeared as a septum 24 h later than the full glucose medium. At 96 h, the cell debris appeared as extracellular riboflavin (Figure 7(h)), which was absent in the full glucose medium and 1 : 4.


## 4. Conclusions

(1)
By using sugarcane molasses as the substrate, four adaptive strains evolved through the adaptation process.A. gossypi A-24 was found to be the most promising in terms of antioxidant and riboflavin production. Under shake flask fermentation, it could adapt to the cells of the strain. The growth and fermentation performance significantly improved. In addition, the effect of cofermentation of different ratios of sugar cane molasses and glucose was studied, and cofermentation with glucose could significantly increase the production of A. gossypii riboflavin and increase the TCA cycle and the carbon flux of the EMP pathway. The adaptability of the strain to molasses environment could be used as a feasible strategy for the efficient and sustainable use of complex agroindustrial waste substrates to produce high-value products.(2)
The best concentration of sugarcane molasses of strain A-24 is 220 g/L. If the concentration is lower or higher than this value, the production of riboflavin will be reduced. The ratio of sugarcane molasses to glucose is 2 : 8, and the yield of riboflavin is the best.



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*Source: 5132198-2022-07-18.xml*