Enhanced production of butyric acid by solid-state fermentation of rice polishings by a mutant strain of Clostridium tyrobutyricum

Purpose: To enhance butyric acid production by solid-state fermentation with a hyper-producing mutant of Clostridium tyrobutyricum generated by random mutagenesis. Methods: Wild type C. tyrobutyricum was mutagenized with UV irradiation, nitrous acid, and ethidium bromide to obtain a hyper-producing strain. Various physiochemical parameters were optimized to increase the butyric acid yield. Results: The UV-induced mutant (C.T) produced significantly higher concentrations of butyric acid than the wild type parent, nitrous acid-induced, and ethidium bromide-induced strains. C.T increased butyric acid production 1.4-fold more than the parent strain. Fermentation with C.T with 2.5 g of rice polishings (w/w), a 2 % inoculum volume (v/v), and a 48-h incubation period at 37°C under anaerobic conditions produced 11.63 mg/100 g of butyric acid. The addition of 0.6 % corn steep liquor as a nitrogen source increased the butyric acid concentration to 26.09 mg/100 g. Conclusion: These optimized fermentation parameters on a small scale can be used on a commercial scale to mass-produce butyric acid.


INTRODUCTION
Butyric acid is an ill-smelling, 4-carbon, short chain fatty acid, CH 3 CH 2 CH 2 COOH, and is an important solvent, polymer, and chemical compound used in the pharmaceutical, food, and chemical industries and is extensively used in thermoplastics.As a raw material, it is used to produce the biodegradable polymer β-hydroxybutyrate [1].It is also used in the treatment of colon cancer and as an energy source for the human body [2], and butyric acid esters give tropical fruits and dairy products their flavors [3,4].Currently, there is great interest in using butyric acid as a precursor to biofuels.
On a commercial scale, butyric acid is produced through an oxy-fuel combustion process with Trop J Pharm Res, July 2018; 17 (7): 1236 petrochemicals as substrates [5].The manufacturing of renewable chemicals by microbes has been stimulated by increases in oil prices, petrochemical industry environmental pollution, and consumer interest in the inclusion of natural ingredients in cosmetics, foods, and pharmaceuticals [6][7][8][9][10].Although numerous Gram-positive obligate anaerobes, such as Clostridium spp.and Butyrivibrio fibrisolnes, produce significant amounts of butyric acid during fermentation [11], Clostridium strains are favored in industry because of their higher outputs and final-product concentrations [12].Clostridium tyrobutyricum is one of the most preferred strains due to its high and stable production of butyric acid [13][14][15].
The cost to produce butyric acid by fermentation depends on the cost of the substrate, and it is costly to use pure carbon sources as substrates.The use of economically feasible and renewable substrates, such as agricultural products, as substrates has been investigated [16].Agroindustrial wastes that are produced in bulk during milling include rice polishings, wheat bran, and molasses.This is the first study that utilized rice polishings for butyric acid production by solid-state fermentation (SSF).More common processes for butyrate production are batch, fed-batch, continuous, and cell-recycle fermentations, but SSF is more economical due to low water requirements, reduced downstream processing, and low stirring requirements [17].
Due to the interest in using butyric acid as a biofuel precursor, this study aimed to hyperproduce butyric acid by creating a stable C. tyrobutyricum mutant and by optimizing fermentation parameters.

Bacterial strain and medium
The parent strain C. tyrobutyricum DSM 2637 (ATCC 25755 NCIB 110635) was procured from DSMZ, Germany.The organism was revived and maintained under anaerobic conditions on Reinforced Clostridial Medium (RCM) agar plates and slants.

Random mutagenesis
C. tyrobutyricum DSM 2637 was exposed to various physical and chemical mutagens to create a mutant with enhanced butyric acid production.

UV irradiation
One mL of a fresh C. tyrobutyricum culture was transferred to RCM agar plates, and the plates were exposed to UV irradiation (Model: Mineral light UVS-12, California, USA) for various amounts of time (10-60 min).By adjusting the distance between the UV lamp and the culture plate to 5.0 cm, a death rate of 95% was achieved [18].
To avoid photostimulation, the UV irradiated cells were grown in the dark in an anaerobic chamber at 37°C for 48 h, colonies were counted, survival curves were generated, and colonies were screened for butyric acid production [15].

Nitrous acid (NA) treatment
NA solutions of various concentrations were prepared by adding 0.9 mL of 0.1 M acetate buffer (pH 6.8) to 0.1 mL of varying concentrations of sodium nitrite (1, 2, 3, 4, or 5 M).Cell pellets of freshly grown 18 h cultures of C. tyrobutyricum were washed with 0.2 M phosphate buffer pH 7.0 and incubated with NA for various amounts of time (20,40, or 60 min) at 37°C [19].The cells were then centrifuged and the cell pellets were washed with phosphate buffer to remove traces of the mutagen.
After washing, the cell pellets were resuspended in 1 mL of 0.2 M phosphate buffer pH 7.0, and serial dilutions were prepared and spread onto RCM agar plates.The plates were incubated in an anaerobic chamber at 37°C for 48 h, colonies were counted, and survival curves were generated.Plates with a death rate >90% were selected and colonies were screened for butyric acid production [15].

Ethidium bromide (EtBr) treatment
Cell pellets of freshly grown 18 h cultures of C. tyrobutyricum were resuspended in 2 mL of saline solution and incubated with varying concentrations of ethidium bromide (15,18,21,24, 27, or 30 mg/mL) at 37°C for 1 h.The cells were centrifuged at 10,000 rpm for 5 min, resuspended in 1 mL of 0.2 M phosphate buffer pH 7.0, and serial dilutions were prepared and spread onto RCM agar plates.
The plates were incubated in an anaerobic chamber at 37°C for 48 h, colonies were counted, and survival curves were generated.Plates with a death rate >90% were selected and colonies were screened for butyric acid production [15].

Fermentation of wild type and mutant C. tyrobutyricum
Single wild type and mutant C. tyrobutyricum colonies were transferred from fresh RCM agar plates to 250 mL Erlenmeyer flasks containing 50 mL of RCM broth and incubated at 37°C for 24 h with shaking at 200 rpm.The cultures were diluted to an OD 600 of 0.6 with RCM medium, added to fermentation media, and incubated under anaerobic conditions at 37°C for 72 h.The fermentation medium contained 0.25% (v/w) NaCl, 0.25% CaCl 2 , 0.3% MgSO 4 , 0.15% KH 2 PO 4 , 0.5% corn steep liquor, and 2.5 g rice polishings at pH 6.0.

Quantification of butyric acid
Quantification of butyric acid was carried out by the organic analysis method [20], which is based on the catalytic oxidation of butyric acid into diacetic acid, which gives a red color upon the addition of sodium nitroprusside.Five mL of a fermented liquor sample was shaken first with 5 mL of a hydrogen peroxide solution, then shaken with one mL of a 5 % ferrous ammonium sulphate solution, followed by shaking with 10 mL of a 10 % sulfuric acid solution.The mixture was heated for 5 min in a water bath at 68 -70 °C.Then, six drops of 20 % sodium hydroxide were added to the cooled mixture and the mixture was filtered.Three drops of a 20% sodium hydroxide solution, three drops of a 5 % sodium nitroprusside solution, and 0.5 mL of acetic acid were added to 5 mL of filtrate.In the presence of butyric acid, a rosy red color is produced, which can be measured spectrophotometrically by the absorbance at 390 nm.The Pearson correlation coefficient was used to evaluate the relationship between OD 390 and butyric acid concentration and showed that there is a positive linear correlation between OD 390 and butyric acid concentration.

Statistical analysis
Data were analyzed statistically using SPSS 16.0 software and were presented as mean ± SD of three replicates Means were compared by One-Way ANOVA, descriptive analysis, and least significant difference (LSD).P < 0.05 were considered significant.

Effect of physical and chemical mutagens on wild type C. tyrobutyricum
After exposing wild type C. tyrobutyricum to UV irradiation, NA, and ethidium bromide, colonies from plates with a >90 % death rate were selected.The survival curves for each mutagenesis treatment are shown (Figure 1A).Mutant strains were screened for increased  butyric acid production using previously optimized fermentation conditions.The mutant strain generated with UV irradiation (C.T UV ) produced 6.86 mg/100 g butyric acid, the mutant strain generated with ethidium bromide produced 3.81 mg/100 g butyric acid, and the mutant strain generated with NA produced 4.73 mg/100 g butyric acid, whereas the wild type strain produced 5.49 mg/100 g butyric acid (Figure 2, Table 1).

Effect of various physiochemical parameters on butyric acid production
We previously determined that rice polishings (2.5 g) are an optimal carbon source for wild type C. tyrobutyricum fermentation, thus we used rice polishings as the carbon source while optimizing other parameters, including the substrate:water ratio, the incubation period, and the inoculum volume, during SSF of the C.T UV mutant strain at 37°C and pH 6.0.Of the various substrate:water ratios, the 10:36 substrate:water ratio gave the highest butyric acid yield of 7.47 mg/100 g (Figure 3B), and of the various incubation periods, 3 days of incubation gave the highest butyric acid yield of 6.86 mg/100 g (Figure 3A).Of the various inoculum volumes tested, 2.5 ml of inoculum gave the highest butyric acid yield of 7.69 mg/100 g, whereas an increase in the inoculum to 3 mL reduced the butyric acid yield to 4.61 mg/100 g (Figure 3C).Various concentrations of NaCl, CaCl 2 , MgSO 4 , and KH 2 PO 4 were tested to determine optimal salt concentrations for the highest butyric acid yield during SSF of rice polishings by the mutant C.T UV at pH 6.0 and 37°C.The salt concentrations of 0.25% (v/w) NaCl, 0.25% CaCl 2 , 0.3% MgSO 4 , and 0.15% KH 2 PO 4 yielded the highest butyric acid concentrations of 8.8 mg/100 g, 9.6 mg/100 g, 10.52 mg/100 g, and 10.66 mg/100 g, respectively (Figure 4 A-D).Various nitrogen:carbon ratios were investigated for maximum butyric acid production.The highest butyric acid yield of 11.63 mg/100 g was observed with a nitrogen:carbon ratio of 1:25 (Figure 5A).Various concentrations of corn steep liquor were also tested for maximum butyric acid production, and 0.6% corn steep liquor gave the highest butyric acid yield of 26.09 mg/100 g (Figure 5B).

DISCUSSION
In this study, we created three C. tyrobutyricum mutants by random mutagenesis, a UV-induced mutant, a nitrous acid-induced mutant, and an ethidium bromide-induced mutant.The UVinduced mutant produced significantly higher concentrations of butyric acid when compared to the ethidium bromide-induced and nitrous acidinduced mutants, and therefore the UV-induced mutant C.T UV was used in further studies.It has been reported that exposure of C. tyrobutyricum to heavy-ion irradiation with 12 C 6+ led to the generation of a C. tyrobutyricum mutant that increased butyric acid production from 2.2 g in wild type to 3.3 g in the mutant [21].The mutant C.T UV significantly increased the butyric acid yield after 3 days of incubation and with a 10:36 substrate:water ratio.The volume of the inoculum generally affects the amount of product produced by fermentation.Previous studies found that 50 mL and 100 mL C. tyrobutyricum inoculum volumes increased butyric acid production by 5% (v/v) to 34.2 g/L and 3.87% to 4.3 g, respectively [10,12,22].In this investigation, a significantly higher concentration of butyric acid was produced with a 2.5 % (v/w) C.T UV inoculum, whereas the maximum butyric acid yield with wild type C. tyrobutyricum was observed with a 2 % (v/w) inoculum.
Butyric acid production may also be affected by the nutritional value of the medium.Rich media usually enhance butyric acid production.More butyric acid was produced by C. tyrobutyricum fermented in RCM than in Clostridial Growth Medium [8,23].A medium containing 500 g/L glucose, 25 g/L MgSO 4 , 1.3 g/L MnSO 4 , and 0.6 g/L FeSO 4 has been recommended to increase butyric acid production [21].It has also been shown that a nutrient formulation composed of 30 g/L glucose, 5 g/L yeast extract, 5 g/L peptone, 3 g/L (NH 4 ) 2 SO 4 , 0.6 g/L MgSO 4 , and 0.03 g/L FeSO 4 increased butyric acid production [24].Further, the addition of corn steep liquor with corn fiber hydrolysate as a substrate has been shown to increase the butyric acid yield [25].In this study, we found a C. tyrobutyricum mutant strain that significantly increased butyric acid production 1.4-fold when compared to the wild type C. tyrobutyricum strain.

CONCLUSION
An SSF technique for butyric acid production using rice polishings as the substrate has been developed.The C.T UV mutant increased butyric acid production to 26.09 mg/100 g, which is 1.4fold more than the butyric acid produced by the wild type parent strain, thus this simple and inexpensive method can be used for butyric acid production on a commercial scale.

Figure 3 :
Figure 3: The effects of various parameters on butyric acid production during SSF of rice polishings by the mutant C.T UV at pH 6.0 and 37°C: (A) Incubation period, (B) Substrate:water ratio, (C) Inoculum volume.

Figure 5 :
Figure 5: Effects of the nitrogen:carbon ratio and the corn steep liquor concentration on butyric acid production during SSF of rice polishings by mutant C.T UV at pH 6.0 and 37°C: (A) the nitrogen:carbon ratio, (B) corn steep liquor

Table 1 :
Statistical analysis of mean concentrations of butyric acid produced by mutant C. tyrobutyricum strains c *Means with different superscripts differ significantly (p < 0.05)