
Different bacterial isolates were isolated from maize, Egyptian clover, lemon, zucchini, banana, and spinach plants grown in different localities in Assiut Governorate. The pure cultures of species were made and maintained on nutrient agar (NA) slants at 4ºC and subcultures monthly.
To test the ability of bacterial isolates to produce riboflavin, single colonies were transferred into 5 ml nutrient broth [35]. After 12 h aerobically incubation at 30 ± 1ºC and 200 rpm, 1 ml of these pre cultures were used to inoculate 50 ml modified basal medium contained g/l: 50 glucose, 5 NaNO3, 0.5 MgSO4, 1.5 KH2PO4, 0.5 K2HPO4, 0.0025 ZnSO4 and 1000 ml distilled water, pH 6.5. Fermentation was carried out at 30 ± 1°C on a rotary shaker (200 rpm) for 72 h [36]. The culture was centrifuged in pre weighted tubes at 4,000 xg rpm for 15 min and the supernatant obtained sterilized by membrane filtration, using a membrane of pore size 0.22 mm and was used as the crude riboflavin solution for quantitative determination of extracellular riboflavin. The biomass was rewashed and recentrifuged using distilled water to remove any remaining sugars produced and dried at 40ºC overnight for cell dry mass (CDM) determination.
To investigate the effect of different incubation times for biomass and riboflavin production of bacterial strains, the culture flasks inoculated with bacterial cells were kept for an extended period of 72 h and incubated at 30 ± 1°C. The riboflavin production and optical density at 600nm (OD600) were measured every 6 h.
In the production medium, different concentrations of glycine 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3 g/l were tested. Each concentration was prepared with three replicates and incubated in the same fermentation conditions. Cell growth was determined by the measurement of optical density at 600nm (OD600) by a spectrophotometer and extracellular riboflavin was measured.
The RSM used in the present study is a central composite design (CCD) involving five different factors. Experiments were conducted in a randomized fashion. The CCD contains a total of 56 experiments, 54 factorial experimental runs with 2 additional runs at the center point level to check reproducibility was performed for five investigated parameters; glucose (A), NaNO3 (B), KH2PO4 (C), K2HPO4 (D) and MgSO47H2O (E), were varied at three coded levels [37]. The range and central point value of all the three process variables are shown in Table 1.
xi= Xi– X0/δX (1)
Y= β0+ Σβi xi +Σ βii xi 2+ Σβijxij(2)
Fermentation broth samples were analyzed for cell dry weight, O. D. (600), glucose and riboflavin concentration: Cell growth was determined by the measurement of optical density at 600nm (OD600) by a spectrophotometer. Glucose was determined using anthronesulphuric method [38]. Standard curve of glucose with different known concentrations was prepared. The standard method of estimation of riboflavin is spectrophotometric using T60 UV with a split beam UV visible spectrophotometer and fixed slit of 2 nm. The instrument covers a wavelength range of 190-1100 nm. A 0.8 ml of centrifuged culture broth was mixed with 0.2 ml of 1 M NaOH and neutralized with 1 ml of 0.1 M potassium phosphate buffer (pH 6.0) as modified by Tajima et al. [39]. The amount of riboflavin in the supernatant was measured quantitatively at 444 nm against substrate-free blank. The standard curve was prepared using pure riboflavin.
Eighty five bacterial isolates were recovered from different parts of maize, Egyptian clover, lemon, zucchini, banana and spinach plants. The purified isolates were screened for their ability to produce riboflavin on fermented medium. Only two isolates were selected for further characters based on the highest riboflavin production. The selected bacterial isolates were identified firstly using morphological and biochemical characteristics as illustrated in Table 2. Then molecular identification using phylogenetic analysis of 16S rRNA gene sequences was performed. The sequence of approximately 1137 and 1151 base pairs of bacterial strains ASU 8 and ASU9, respectively have sequence with 99% similarity to Bacillus subtilis (GQ480495) and 99% Bacillus tequilensis (JF411314). So, the bacterial strains were identified as Bacillus subtilis ASU8 (KU559874) and Bacillus tequilensis ASU9 (KU559876). These strains produced the highest amounts of riboflavin on basal medium giving 81.58 ± 0.62, 74.43 ± 0.32 mg/l and dry mass 0.65 ± 0.012, 0.62 ± 0.05 g/l basal medium of Bacillus subtilis and Bacillus tequilensis, respectively and used for further experiments.
Riboflavin production in the filtrate was detected over range of 0-72 h (Figure 1 A-B). The production not started until 18-24 h.; however growth started after 6h. The maximum optical density (OD 600) was 0.981, 0.947 and the maximum riboflavin production was 112.29, 108.95 mg/l by Bacillus subtilis ASU8 (KU559874) and Bacillus tequilensis ASU9 (KU559876), respectively. The riboflavin production of constructed strain RF18S was 1.8-fold higher as compared to that of recombinant strains of B. subtilis in flask batch culture, 387.6 mg L−1 riboflavin versus 220mg L−1 riboflavin [40] and higher than that of A. gossypii in flask batch culture was 228 mg L−1 riboflavin [41]. Lin et al. [42] found that, riboflavin accumulation and a titer of 225.1mg L−1 riboflavin was produced by strain E. coli RF01S. Riboflavinproducing Escherichia coli strain could produced 387.6 mg L−1 riboflavin with a yield of 44.8 mg riboflavin per gram glucose in shake-flask fermentations after over expressing rib B and engineering purine pathway [43]. Sauer et al. [9] reported that a recombinant, riboflavinproducing strain of Bacillus subtilis give productivities of 80 mg/l at 0.3 h-1 using a glucose-limited chemostat. Li et al. [44] found that when the specific growth rate is at 0.27/h, the theoretical specific riboflavin production rate in B. subtilis could reach 0.48 mmol/g CDW/h, Clostridium acetobutylicum, which had a productivity of about 100 mg/l, was one of the first organism used to produce riboflavin [11]. Engineered microbes are able to synthesize large quantities of riboflavin as well as catalyzing the other process. These microbes that incorporate all required functionalities into a single strain have proven inherently challenging. These challenges result from plasmid loss occurring during the fermentation process, fragile and instability of engineered cells. Two major reasons for plasmid loss include plasmid instability due to the segregational plasmid loss during cell division and depression of the growth rate of plasmid bearing cells [45, 46]. These drawbacks limit their use in real world applications like industrial bio processing. In sharp contrast to engineered microbes, natural producing isolates holds many appealing properties in a bio processing context, such as stability, functional robustness and the ability to perform complex tasks [47].
To investigate the effect of the addition of glycine on riboflavin production in Bacillus subtilis ASU8 (KU559874) and Bacillus tequilensis ASU9 (KU559876), glycine added to the culture in the amounts of 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2 and 3 g/l, respectively (Figure 2 A-B). The addition of glycine support and increased the riboflavin production. With the addition of 1 g/l of the glycine, the increase was even 144.74, 184.21mg/l (1.77 %, 2.5%) higher than in control for Bacillus subtilis and Bacillus tequilensis, respectively. Riboflavin production decreased with the increase of the concentration of glycine over 1 g/l. Also it was found that there is a reverse relationship between growth, riboflavin production and glucose concentration on medium, with increasing growth glucose concentration decrease. Nanchen et al. [48] recorded that, varying from 0.11 to 0.38 mmol (glucose) g−1h−1, the substrate maintenance coefficient of E. coli is obviously lower than that of wild-type B. subtilis (0.44 mmol (glucose) g−1h−1, [9], which might contribute to a higher riboflavin yield when a high cell density fermentation process is conducted. Glycine found to be stimulator for riboflavin overproduction as a precursor of purine and was not associated to cell growth, but only product formation [26]. The effect of glycine addition was studied by both A. gossypii [27] and C. famata [28] and the incorporation of 14C-glycine into riboflavin produced by A. gossypii was shown by Plaut [29] who reported that the effect of glycine supplement on productivity could mean that glycine is either only a limiting precursor or additionally an inducer.
By applying multiple regression analysis on the experimental data, the following fitting second-order polynomial equation was found to describe riboflavin production for Bacillus subtilis (Equation 3), Bacillus tequilensis (Equation 4):
Variable | Component | Level of variable (g/l) | ||
-1 | 0 | +1 | ||
A | Glucose | 40 | 50 | 60 |
B | NaNO3 | 3 | 5 | 7 |
C | KH2PO4 | 0.5 | 1.5 | 2.5 |
D | K2HPO4 | 0.1 | 0.5 | 1 |
E | MgSO4.7H2O | 0.1 | 0.5 | 1 |
Characteristics | ASU 8 Bacillus subtilis (KU559874) |
ASU 9 Bacillus tequilensis (KU559876) |
Shape | bacillus | bacillus |
Spore formation | + | + |
Motility test | + | + |
Gram’s staining | + | + |
Ryu’s method | watery | watery |
Catalase test | + | + |
Oxidation-Fermentation test | - | - |
Nitrate reductase | + | + |
Gelatin hydrolysis | + | + |
Casein hydrolysis | + | - |
Starch hydrolysis | + | + |
Indole production | - | + |
Arginine dihydrolase | - | + |
Urease test | - | - |
H2S production | - | - |
Voges-Proskauer | + | - |
Carbon source utilization: | + | |
L-Arabinose | - | - |
D-Fructose | + | + |
Citrate | + | - |
D-Sorbitol | - | - |
Sorbose | + | - |
D-Galactose | + | - |
Glycerol | + | + |
Glucose | + | + |
Sucrose | + | + |
Lactose | + | - |
Maltose | + | + |
Mannitol | + | + |
Growth at 4°C | - | - |
Growth at 37°C | + | + |
Growth at 41°C | + | + |
Factor | Coefficient | t value | P value | |||
B. subtilis | B. tequilensis | B. subtilis | B. tequilensis | B. subtilis | B. tequilensis | |
Constant | 174.3 | 195.03 | 83.0 | 77.89 | 0.0000 | 0.0000 |
A: Glucose | -8.7 | -34.5 | -6.1 | -44.6 | 0.0000 | 0.0000 |
B: NaNO3 | -25.2 | -2.98 | -17.8 | -3.85 | 0.0000 | 0.0005 |
C: KH2PO4 | 7.3 | 7.67 | 5.1 | 9.92 | 0.0000 | 0.0000 |
D: KH2PO4 | 13.2 | 9.04 | 9.3 | 11.71 | 0.0000 | 0.0000 |
E:MGSO4.7H2O | -4.1 | -3.78 | -2.9 | -4.89 | 0.0064 | 0.0000 |
A B | 1.8 | 1.74 | 1.2 | 1.84 | 0.2494 | 0.0739 |
A C | 13.8 | 20.93 | 9.2 | 22.1 | 0.0000 | 0.0000 |
A D | 18.7 | -4.09 | 12.4 | -4.3 | 0.0000 | 0.0001 |
A E | 19.8 | 1.75 | 13.2 | 1.85 | 0.0000 | 0.0726 |
B C | -4.9 | 9.62 | -3.3 | 10.17 | 0.0025 | 0.0000 |
B D | 12.5 | -1.83 | 8.3 | -1.94 | 0.0000 | 0.0611 |
B E | 0.04 | 8.99 | 0.02 | 9.50 | 0.9800 | 0.0000 |
C D | -3.9 | 14.67 | -2.6 | 15.51 | 0.0130 | 0.0000 |
C E | -15.9 | -14.3 | -10.6 | -15.1 | 0.0000 | 0.0000 |
D E | -32.5 | -8.531 | -21.6 | -9.01 | 0.0000 | 0.0000 |
AA | -37.3 | 4.36 | -9.7 | 5.32 | 0.0000 | 0.0000 |
BB | 28.4 | -29.19 | 7.4 | -35.62 | 0.0000 | 0.0000 |
CC | -19.9 | 1.8 | -5.2 | 2.21 | 0.0000 | 0.0336 |
DD | -6.68 | -4.4 | -1.7 | -5.37 | 0.0916 | 0.0000 |
EE | 37.0 | -7.18 | -8.76 | 9.61 | 0.0000 | 0.0000 |
t – student’s test, p – corresponding level of significance,* Significant at p ≤0.05, N, non-significant at p≥0.05 |
Source | Degree of freedom | Sum of squares | Mean square | F-value | P-value |
Bacillus subtilis (KU559874) | |||||
Model | 20 | 128689 | 6434.4 | 88.631 | 0.0000 |
Error | 35 | 2540.9 | 72.598 | ||
Lack of Fit | 6 | 652.96 | 108.83 | 1.672 | 0.0000 |
Pure Error | 29 | 1888.0 | 65.102 | ||
Total (Model + Error) | 55 | 131230 | 2386.0 | ||
Bacillus tequilensis (KU559876) | |||||
Model | 20 | 150631 | 7531.5 | 62.77 | 0.0000 |
Error | 35 | 1003.2 | 28.662 | ||
Lack of Fit | 6 | 552.86 | 92.143 | 5.934 | 0.0004 |
Pure Error | 29 | 450.33 | 15.529 | ||
Total (Model + Error) | 55 | 151634 | 2757.0 |

Figure 1: Effect of incubation time on growth and riboflavin production by Bacillus subtilis ASU8 (A) and Bacillus tequilensis ASU9 (B).

Figure 2: Effect of adding glycine on growth and riboflavin production by Bacillus subtilis ASU8 (A) and Bacillus tequilensis ASU9 (B).

Figure 3: Comparison between riboflavin production (mg/l) experimental and predicted values of the RSM model by Bacillus subtilis ASU8 (A) and Bacillus tequilensis ASU9 (B).

Figure 4A1: Response surface plots of riboflavin production by Bacillus subtilis showing the effect of two variables (other variables were kept at zero in coded unit): significant interaction between A1: Glucose and KH2PO4.

Figure 4A8: K2HPO4 and MgSO4.7H2O for Bacillus subtilis ASU8.
Response surface plots of riboflavin production by Bacillus subtilis & Bacillus tequilensis showing the effect of two variables
(other variables were kept at zero in coded unit): significant interaction between

Figure 4B1: Response surface plots of riboflavin production by Bacillus tequilensis showing the effect of two variables (other variables were kept at zero in coded unit): significant interaction between
B1: Glucose and KH2PO4.
- Dmytruk K., et al. “Construction and fed-batch cultivation of Candida famata with enhanced riboflavin production”. Journal of Biotechnology 172(2014): 11– 17.
- Bodiga VL., et al. “Effect of vitamin supplementation on cisplatin-induced intestinal epithelial cell apoptosis in Wistar/NIN rats”. Nutrition 28.5 (2012): 572-580.
- Mal P., et al. “Azithromycin in combination with riboflavin decreases the severity of Staphylococcus aureus infection induced septic arthritis by modulating the production of free radicals and endogenous cytokines”. Inflammation Research 62.3 (2013): 259-273.
- Basu TK and Dickerson JWT. In Cab International (Ed.), Vitamins in human health and disease. United Kingdom: Wallingford. (1996).
- Moat S J., et al. “Effect of riboflavin status on the homocysteine-lowering effect of folate in relation to the MTHFR (C677T) genotype”. Clinical Chemistry 49.2 (2003): 295-302.
- Wacker J., et al. “Riboflavin deficiency and preeclampsia”. Obstetrics and Gynecology 96.1 (2000): 38-44.
- Lane M and Alfrey CP. “The anemia of human riboflavin deficiency”. Blood 25.4 (1965): 432-442.
- Arbige MV., et al. Fermentation of Bacillus, p. 871–895. In A. L. Sonenshein., et al. Bacillus subtilis. American Society for Microbiology, Washington, D.C. (1993)
- Sauer U., et al. “Physiology and metabolic fluxes of wild-type and riboflavin producing Bacillus subtilis. Microbiology 62.10 (1996): 3687-3696.
- Kurth R., et al. Riboflavin. pp. 521-530. Ullmann’s Encyclopedia of Industrial Chemistry. Weinheim, Germany: Wiley-VCH. (1996).
- Lim SH., et al. “Microbial production of riboflavin using riboflavin overproducers, Ashbya gossypii, Bacillus subtilis, and Candida famata: an overview”. Biotechnology and Bioprocess Engineering 6 (2001): 75-88.
- Stahmann KP., et al. “Three biotechnical processes using Ashbya gossypii, Candida famata or Bacillus subtilis compete with chemical riboflavin production”. Applied Microbiology and Biotechnology 53.5 (2000): 509-516.
- Vandamme EJ. “Production of vitamins, coenzymes and related biochemicals by biotechnological processes”. Journal of Chemical Technology and Biotechnology 53.4 (1992): 313-327.
- Lee KHK., et al. Microorganism for producing riboflavin and method for producing riboflavin using the same, European patent EP1426446 B1. (2008).
- Schallmey M., et al. “Developments in the use of Bacillus species for industrial production”. Canadian Journal of Microbiology 50.1 (2004): 1-17.
- Burgess CM., et al. “Bacterial vitamin B2, B11and B12 overproduction: an overview”. International Journal of Food Microbiology 133.12 (2009): 1-7.
- Perkins JB., et al. “Riboflavin overproducing strains of bacteria”. European patent application 0-405-370-A1 (1991).
- Bacher A., et al. “Biosynthesis of riboflavin”. Vitamins and Hormones 61(2001): 1-49.
- Ghozlan HA. “Utilization of beet molasses for riboflavin production by Mycobacterium pheli”. 34.3 (1994): 157-162.
- Humbelin M., et al. “GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production”. The Journal of Industrial Microbiology and Biotechnology 22.1 (1999): 1-7.
- Foor F and Brown GM. “Purification and properties of guanosine troposphere cyclohydrolase II from Escherichia coli” Journal of Biological Chemistry 250.9 (1975): 3545-3551.
- Bresler SE and Perumov DA. “Study of the operon for riboflavin biosynthesis in Bacillus subtilis. Influence of genotype on regulating the synthesis of GTP-5- triphosphate cyclohydrolase”. Genetika 15 (1979): 967-971.
- Shavlovskii GM., et al. “Determination of GTP-cyclohydrolase, the first enzyme of riboflavin biosynthesis from the yeast Pichia guilliermondii”. Doklady Akademii Nauk SSSR 230 (1976): 1485-1487.
- Shavlovskii GM., et al. “Regulation of synthesis of GTP-cyclohydrolase participating in yeast falvinogenesis by iron”. Microbiologia 46.3 (1977): 578-580.
- Stryer L., et al. “Biosynthesis of nucleotides”. Biochemistry 4th edition (1995): 739-740.
- Kaplan L and Demain AL. “Nutritional studies on riboflavin overproduction by Ashbya gossypii”.(1970): 137-159. In: D. G. Ahearn (edition). Recent Trends in Yeast Research. Georgia State Univ., Atlanta, GA, USA.
- Hanson A M. “Microbial production of pigments and vitamins”. In: Peppler HJ (edition) Microbial technology. Reinhold, New York, (1967): 222-250.
- Heefner D., et al. “Riboflavin producing strains of microorganisms, method for selecting, and method for fermentation”. Patent WO 88/09822(1988).
- Plaut GWE. “Biosynthesis of ribofavin. II. Incorporation of 14C-labelled compounds into ring A”. Journal of Biological Chemistry 211 (1954): 111-116.
- Guo WQ., et al. “Optimization of culture conditions for hydrogen production by Ethanoligenens harbinense B49 using response surface methodology”. Bio Resource Technology 1003 (2009): 1192-1196.
- Venkata Dasu V., et al. “Development of medium for griseofulvin production: part II. Optimization of medium constituents using central composite design”. Journal of Microbiology and Biotechnology 12.3 (2002): 360-366.
- Bezerra MA., et al. “Response surface methodology (RSM) as a tool for optimization in analytical chemistry”. Talanta 76.5 (2008): 965-977.
- Brenner DJ., et al. “Bergey’s manual of systematic bacteriology, vol. 2”. The Proteobacteria East Lansing USA 183 (2005).
- Ausubel FM., et al. “Current protocols in molecular biology”. New York, NY: J. Wiley and Sons. (1998).
- Atlas RM. “Handbook of Microbiological Media”. Chemical Rubber Company (1993).
- Shi S., et al. “Increased production of riboflavin by metabolic engineering of the purine pathway in Bacillus subtilis”. Journal of Biomedical Engineering 46.1(2009): 28-33.
- Myers RH and Montgomery DC. “Response Surface Methodology: Process and Product Optimization Using Designed Experiments, first edition”. John Wiley & Sons New York. (1995).
- Halhoul M and Kleinberg I. “The Differential determination of glucose and fructose, and glucose-and fructose-yielding substances with anthron”. Analytical biochemistry 50.2 (1972): 337-343.
- Tajima S., et al. “Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii”. The Journal of Bioscience and Bioengineering 108.4 (2009): 325-329.
- Shi SB., et al. “Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production”. Metabolic Engineering 11.4-5 (2009): 243-252.
- Jimenez A., et al. “Phosphoribosyl pyrophosphate synthase activity affects growth and riboflavin production in Ashbya gossypii”. BMC Biotechnology 8 (2008): 67.
- Lin ZQ., et al. “Metabolic engineering of Escherichia coli for the production of riboflavin”. Microbiology Cell Factories 13 (2014): 104.
- Xu Z., et al. “Improvement of the riboflavin production by engineering the precursor biosynthesis pathways in Escherichia coli”. The Chinese Journal of Chemical Engineering 23.11 (2015):1834-1839.
- Li XJ., et al. “Redirection electron flow to high coupling efficiency of terminal oxidase to enhance riboflavin biosynthesis”. Applied Microbiology and Biotechnology 73.2(2006): 374-383.
- Anderson TF and Lustbader E. “Inheritability of plasmids and population dynamics of cultured cells”. Proceedings of the National Academy of Sciences 72.10 (1975): 4085-4089.
- Shin HS and Lim HC. “Optimal fed-batch operation of recombinant cells subject to plasmid instability and death”. The journal Bioprocess and Biosystems Engineering 31.6 (2008): 655-665.
- Mee MT and Wang HH. “Engineering ecosystems and synthetic ecologies”. Molecular BioSystems 8.10 (2012): 2470-2483.
- Nanchen A., et al. “Non linear dependency of intracellular fluxes on growth rate in miniaturized continuous cultures of Escherichia coli”. Applied and Environmental Microbiology 72 (2006): 1164-1172.
- Nafady NA., et al. “Improvement of medium components for high riboflavin production by Aspergillus terreus using response surface methodology”. Rendiconti Lincei 26.3 (2015): 335-344.