Microbial Biotransformation of Agro-waste Into Biovanillin as Flavor: A Process Optimization by Response Surface Methodology

Vanillin is a flavour that is commonly employed in the food industry, but plant sources can yield a small amount of this molecule. Fermentative production of biovanillin by optimizing different process parameters such as specific pH, temperature, incubation time, carbon source, nitrogen source, ferulic acid substrate, and by agro wastes are beneficial in terms of fermentation economics and scale up of the process response surface methodology for an ideal production of biovanillin yield. Hence the present research emphasizes on optimization of fermentation conditions for employing Bacillus licheniformis MSJM 5 isolate. As per the experimental results biovanillin production yield was enhanced at neutral pH, mesophilic temperature, in an incubation of 48 hrs, under revolving conditions, in the presence of glucose as carbon source soya bean as nitrogen source ferulic acid 0.3 mg concentration and ground nut oil cake, an agro waste material has stimulated the formation of biovanillin, 326-834 mg/ml. Further stastical analysis by RSM the yield of biovanillin was 1.89 mg/ml. Based on the morphological cultural and biochemical the positive strain MSJM 5 is identified as Bacillus licheniformis with 98% homology and accession number ON413745. Strain improvement studies have reveated that UV light and Et Br use reduce the survival capacity of Bacillus licheniformis .


Introduction
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an essential organoleptic component of the vanilla flavour and is considered a secondary plant metabolite (Galadima et al., 2020).Physico chemically,it is a white crystalline powder with a sweet vanilla-like smell.It is an aromatic aldehyde that belongs to the group of simple phenolic compounds with the molecular formula of C 8 H 8 O 3 (Mehmood et al., 2022).Its functional groups are phenol, aldehyde, and ether.It is primarily used in the beverages, perfumes, medical, pharmaceutical, and food industries because of its antioxidant and antimicrobial properties (Sujatha et al., 2022).In pharmaceutical industry, it is used as suppress the unpleasant taste and odor of medicine.
Biovanillin refers to as vanillin procured through the biotechnology process using natural precursors like eugenol, isoeugenol, guaiacol, ferulic acid (FA), phenolic stilbenes, aromatic amino acids creosol, sugars, vanillic acid, vanillyl amine, and waste residues used to bio transform by microorganisms or by isolated enzymes.Ferulic acid is an ideal precursor for biovanillin production due to its structural similarity to vanillin (Saeed et al., 2021).FA (4-hydroxy-3-methoxycinnamic acid) is an excellent natural aromatic product in plants, and it occurs as a constituent of cell walls in many agriculture seed, so the valorization of agricultural waste can be cost-effective as commercial FA is expensive (Chakraborty et al., 2017).Ferulic acid is present in large amounts in corn hulls (31.0 g/kg) followed by maize bran (30 g/kg),sugar beet (5-10 g/kg), and wheat barley grains containing 6.6 and 1.4 g/kg, respectively.Ferulic acid is released when digested with alkali or by using enzymes such as ferulic acid esterase and cinnamoyl esterase used in conjunction with glycosyl hydrolases present in the plant cell wall (Mazhar et al., 2017).Various fruit peel by-products such as pomegranate, banana, and orange were evaluated to obtain FA and the reported concentrations were 1.55 mg/g (Asrabi et al., 2016), 0.61 mg/g, and 0.339 mg/g FA, respectively (Myers et al., 2020).
Confidencing the significance of the agro waste use and microbial production of vanillin of agriwaste the current study was focused on the optimization of biovanillin production using response surface methodology (RSM) design.The central composite design (CCD) of RSM provides an understanding of the interaction between different parameters and to identify the bacterial isolate MSJM 5 at molecular level.

Optimization of Culture Conditions for Biovanillin Production
To produce biovanillin the cultural conditions like static and shaking condition, incubation time, pH, temperature, carbon, nitrogen sources, ferulic acid and agricultural waste were optimized.After incubation of MSJM 5 in amedium the culture filtrates were collected in a medium centrifuged and then used for the estimation of biovanillin.

Effect of pH
The influence of pH on the biovanillin production was determined at various pH ranges from 5 to 9 in a buffer of phosphate (0.1 M) and inoculated in basal media.Each media along with MSJM 5 inoculam were incubated at 37 °C for 24 hrs in orbital shaking condition (150 rpm).

Effect of Temperature
Temperature optimization was done by incubating 100 ml of modified media composed of 0.5% of ferulic acid as a substrate of temperatures ranged from 20 to 70 °C.After 24 hours of incubation of MSJM5 the medium was harvested for estimation of biovanillin.

Effect of Incubation Time
The incubation time for MSJM 5 inoculum was optimized by incubating the strain for different time intervals of 0, 2, 4, 8, 12, 24, 48 and 72 hrs.After incubation the biovanillin production was calculated from harvested medium by using UV-visible spectrum analysis (Kaur et al., 2013).

Effect of Ferulic Acid Substrate
The effect of ferulic acid as substrate was evaluated by adding different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 mg/ml) in 100 ml of prepared ferulic acid into minimal media and the potent strain was inoculated and incubated at 37 °C for 24 hrs.After incubation the biovanillin production was measured and calculated.The vanillin produced in any condition of medium studied here was estimated using UV, VIS spectrophotometry even if not menctioned.

Effect of Carbon Sources
One gram of each carbon source was mixed with 100 ml of medium such as glucose 1 gm, sucrose 1 gm, maltose 1 gm, mannitol 1 gm, fructose 1 gm were used as sole source for experiments in triplicates and mean values were represented.

Effect of Organic Nitrogen Source
Different organic nitrogen sources of 1 g of soya meal, malt extract, yeast extract, peptone and gelatin were added in 100 ml of basal media inoculated and incubated at 37 °C for 24 hrs (Paz et al., 2013).

Effect of Agrowastes on Biovanillin Production
Submerged fermentation was carried out on a variety of substrates, including wheat bran, soya bean meal, rice bran, sesame oil cake, groundnut oil cake, coir pith, mixed fruit and orange peel.For the formation of biovanillin, 5 grams of substrate were introduced separately to the 100 ml basal medium.Fermentation was performed using a 2% inoculum.The medium was incubated for 32 hours at 37 °C.

Statistical Analysis
All of the tests were done in triplicates, and the results were calculated as a mean standard deviation with a 95 percent confidence interval (p ≤ 0.05) using data analysis software (Graph Pad Prism, version 5.0.1,San Diego, CA).

Optimization of Medium Components for Biovanillin Production Using Response Surface Methodology (RSM)
The interactions between the medium components on biovanillin production were studied by response surface methodology (RSM) using Central composite design.

Identification of MSJM 5 Strain, Morphological Characterization
The morphological features of selected bacteria were studied by using gram staining, spore staining and hanging drop techniques (Sinha et al., 2008).

Cultural Characterization
Colony morphological features such as size, shape, color, texture and surface margin of the colonies of the selected bacteria on the suitable medium were studied and results were recorded.Characterization (Cheesbrough, 2006) The biochemical characterization studies were done with the following test following standard procedures defined by Cheesbrough (2006)

Molecular Characterization of Positivie Strain by Gene Sequencing of 16SrRNA
The sequencing reactions for isolates were performed in Applied Biosystems (Bioaxis, Hyderabad).Sequences were further used for BLAST analysis from the NCBI data base to obtain the sequence similarity with related organisms.The sequences were multiple aligned with representative sequences of selected genera using CLUSTAL W program.The phylogenetic tree for the datasets was utilized to access the relationship between the organisms using the PHYLIP analysis programme.The sequences obtained were deposited in Gen Bank and accession number was obtained.

Strain Improvement of the Potent Microbial Isolate for Increase in Biovanillin Production Physical Mutagenesis
Ten ml of microbial inoculum diluted suspension was transferred to the sterilized petriplates.The petriplates were then placed under the UV lamp at an intensity of 254 µw/cm 2 for 5 min-2 h time duration.After each 30 min time interval 0.5 ml of the bacterial suspension was transferred to the petriplates containing nutrient agar medium.The plates were then placed for incubation at 37 °C for 24 hours, to detect mutant colony morphologicaly.

Chemical Mutagenesis
A stock solution having 0.5 mg/mL (w/v) concentration of ethidium bromide (Et Br) was prepared and 1.0 mL of it was added to 9.0 mL of nutrient agar media containing colonies of MSJM 5 .After specific time intervals of 5 min-2 h minute incubation (30 min interval), it was centrifuged three times at 10,000 rpm for 15 min.After treating the colonies with Et Br 100-fold serial dilutions was done to visualize the chemical mutagen resistant colonies (Averesch et al., 2014).

Effect of pH on Biovanillin Production
The effect of various pH levels for the production of biovanillin by MSJM5 strain was analysed by growing the organism in media with different pH values of 5 to 9. pH 7.0 was found to ideal for the biovanillin maximum production (Figure 1).Among different pH levels, pH 7.0 was shown highest yield 326 mg/ml.jas.ccsenet.

Effec
The effect media at d was grown strain MSJ   runs.ment bined may be addressed.In the second order model, the generally utilised response surface design (CCD) involves 5 stages for each element required for quadratic terms to be estimable.The F-value of 4.65 for the model indicates that it is significant (Tables 1 and 2).An F-value of this magnitude has a 0.98 % probability of occurring due to noise.Model terms are important when "Prob > F" is less than 0.0500.B and BC are important model variables in this scenario.If the value is more than 0.1000, the model terms are irrelevant.If your model has a large number of insignificant model terms, model reduction may improve it (not including those necessary to maintain hierarchy).The "Lack of Fit F-value" of 0.88 suggests that the lack of fit has no influence on the purely erroneous result.There's a 58.52% likelihood that a Noise causes a significant "Lack of Fit F-value".If there's a minor mismatch, that's fine; we want the best fit possible.As a disadvantage, the term "Pred R-Squared" implies that the overall mean may be a more accurate predictor of your answer than the current model."Adeq Precision" is used to calculate the signal-to-noiseratio.It is better to have a four-to-one ratio.You have a good signal with a signal-to-noise ratio of 10.145.This concept can assist you in navigating the design world.
jas.ccsenet.The molecular characterization of MSJM 5 stain was carried out using the 16SrRNA gene sequence analysis.
Nearly full length 16SrRNA gene was amplified and sequenced using 16SrRNA universal primers.When subjected to BLAST analysis by using NCBI BLAST similarity search tool, the resultant 16SrRNA sequence data of MSJM 5 was 98% homologous with the sequence data of Bacillus licheniformis obtained from the nucleotide data base of National Centre for Biotechnology Information (NCBI)-Genbank9 (Flat File) (Figure 11).A consensus 94 bootstrapped phylogenetic tree was constructed by neighbour joining method with 1000 replicates using MUSCLE 3.7 program in order to evaluate the evolutionary relation of our strains of interest.Finally, MSJM 5 sequence data was submitted in NCBI Genbank under the accession number with ON413745 the unique strain name Bacillus licheniformis (Figure 4).The potent isolate MSJM 5 was isolated, characterized and identified by different morphological, cultural, biochemical and molecular characterization techniques.The colonies of MSJM 5 appeared flat, irregular, rough and yellow in colour with undulate margin.The strain MSJM 5 was motile.Upon gram staining they appeared as +ve rods.Standard biochemical tests were carried out for the strain MSJM 5 .The organism was found to be positive for substrates glucose, fructose, sucrose, and maltose.This organism was able to hydrolyze starch, and gelatin too.The strain MSJM 5 is indole -ve, methyl red +ve, VP +ve.
The strain was able to utilize citrate and unable to produce urease.Finally, the strain MSJM 5 was subjected to 16SrRNA sequence analysis.Phylogenetic tree was constructed based on consensus sequence using nearest neighbor joining method.The organism was finally identified as Bacillus licheniformis and endowed with Gen Bank accession number ON413745 Bacillus licheniformis.Even though species of Bacillus producing biovanillin is evident, Bacillus licheniformis is not reported in the available literature.Several species of fungi and yeast as well as bacterial species Pediococcus are also found to release vanillin from ferulic acid. jas.ccsenet.

Fig
Figure 12 diation on biov

Discussion
Significant increase in biovanillin production by MSJM 5 strain was noticed at both pH 7, 326 mg/l and pH 8, 300 mg/l but compartively more at pH 7. At acidic pH of 5 min, minimum amount of biovanillin was formed 113 mg/l.Veena Paul et al. ( 2021) studied on process optimization for enhanced vanillin production using Bacillus aryabhatai NCIM5503 and demonstrated that pH 8 and 8.5 are more favorable for biovanillin production (192 to 195 mg/l) followed by pH 7.2 with an yield of 6.4 mg/ml, like use Saaed et al. ( 2021) identified 3.5 to 4.5 biovanillin recovery at pH 7. The effect of incubation period on the production of biovanillin by MSJM 5 was evaluated by incubating the culture medium for different time periods viz., 1, 4, 8, 12, 24, 48 and 72 hours.The incubation period of 48 hrs was found to be ideal for maximum growth and resulted in maximum biovanillin production at 48 hrs of incubation.In a study conducted by Gnanasekaran et al. (2016) maximum biovanillin production 624 mg/ml at 72 hrs of incubation period was found in white rod fungi Phanerochaete chrysosporium.
An increasing trend in biovanillin production was observed with increasing incubation period.However my research work using MSJM 5 strain on biovanillin production has reduced the incubation period than the earlier report.
Optimum temperature for the production of biovanillin was determined by incubating the MSJM 5 culture at different temperatures viz., 25,30,35,40,45 and 50 o C. The temperature 35 o C was found to be ideal for the production of biovanillin.The bio vanillin production of the MSJM 5 at temperature 37 o C was found to be 417 mg/ml.Braga et al. (2018) have reported maximum bio vanillin production by Enterrobacter hormaechei at 30 o C. As per the study maide by Chen et al. (2016), the result indicated maximum conversion efficiency of 72% ferulic acid into biovanillin at 35 o C, whereas efficiency decreased significantly when the temperature was above 35 o C.This result indicates that the temperature is an important factor for the energy dependent mechanisam in ferulic acid bioconversion by microbial cells.
The carbon source for the optimal production of biovanillin by the strain MSJM 5 was determined by culturing the organisms using the sources glucose, fructose, maltose, sucrose and maltose.The strain MSJM 5 was grown in basal media amended with 1% of carbon sources, Maximum biovanillin production was observed in the strain grown on medium amended with glucose as the carbon source.In this case the biovanillin production was found to be 589 mg/ml.Ding et al. (2015) reported maximum production of biovanillin yield by Streptomyces setonii, when grown in culture medium amended with glucose.In early report Bacillus aryabhatai formed 2.4 g/l vanillin concentraction with glucose as carbon source and 0.71 g/l yield was found with fructose.
The impact of nitrogen on the production of biovanillin was determined by growing the MSJM 5 strain in basal medium amended with 1% organic nitrogen sources such as yeast extract, peptone, soya meal, malt extract, peptone and gelatin independently or separately.Among the different nitrogen sources, soya bean meal resulted in maximum production of biovanillin by the strain MSJM 5 .In this case 498 mg/ml of biovanillin production was observed.Eudes et al. (2014) reported maximum production 235 mg/ml of biovanillin by Streptomyces setonii, when the medium was amended with 1% of soya bean meal.After observation of MSJM 5 growth at optimal pH, temperature substrate, carbon source, nitrogen source and agro-waste for production of biovanillin the MSJM 5 product was analyzed and characterized.
Although the bioconversion of ferulic acid to biovanillin by several micro-organism was intensely studied, only few papers described the use of ferulic acid obtained from agro-industrial wastes and, in particular, no work has been reported with MSJM 5 .In addition, an applicable process for ferulic acid extraction from biomass material has not yet been developed, although this phenolic compound occurs widely in plant world (Rita et al., 2021).In this respect, present study aims to develop a protocol for the pre-treatment of wheat bran and the enzymatic release of ferulic acid from it and to test the suitability of the previously developed protocol for the production of vanillin from ferulic acid obtained by rice bran hydrolysis.
Response surface methodology is one of the most use full techniques used for the optimization of medium components and other factors responsible for the production of biovanillin (Chen et al., 2016).In the present study, the optimal media conditions for bio vanillin production by MSJM 5 strain were obtained by the Central Composite Design (CCD).Zamzuri et al. (2014) reported that the successful optimization of a culture medium for the production of bio vanillin by Bacillus licheniformis.In this study, the optimum concentrations of the most significant factors (glucose, soya bean meal and ferulic acid) for the production of the bio vanillin from potential MSJM 5 was obtained from the Central composite design (CCD) and analyzed by RSM.
The potent isolate MSJM 5 was isolated, characterized and identified by different morphological, cultural, biochemical and molecular characterization techniques.
The colonies of MSJM 5 appeared flat, irregular, rough and yellow in colour with undulate margin.The strain MSJM 5 was motile.Upon gram staining they appeared as +ve rods.Standard biochemical tests were carried out for the strain MSJM 5 .The organism was found to be positive for substrates glucose, fructose, sucrose, and maltose utilization.This organism was able to hydrolyze starch, and gelatin too.The strain MSJM 5 is indole -ve, methyl red +ve, VP +ve.The strain was able to utilize citrate and unable to produce urease.Finally, the strain MSJM 5 was subjected to 16SrRNA sequence analysis.Phylogenetic tree was constructed based on consensus sequence using nearest neighbor joining method.The organism was finally identified as Bacillus licheniformis and endowed with Gen Bank accession number ON413745 Bcillus licheniformis.Even though species of Bacillus producing biovanillin is evident, Bacillus available is not reported in the available literature.Several species of fungi and yeast as well as bacterial species Pediococcus are also found to release vanillin from ferulic acid.
As there is no evidanec of strain improvement studies in early report an attempt has been made to improve the MSJM 5 strain for increased production of biovanillin by UV irradiation (5-2 h) and ethidium bromide Et Br.
(5-30 m), exposure for uv irradiation survival was noiced up to 2 h.whereas with chemical mutagenesis scanty or visibley no growth was noticed .Significant morphological change was noticed with physical mutagenesis but no improvement in the biovanillin yield.Hence the mutants were not used for optimization studies.

Figure
Figure

Figure
Figure 2. E Figure 11.M . Catalase test, Oxidase test, Glucose fermentation test, Starch hydrolysis test, Gelatin hydrolysis test, Nitrate reduction test, H 2 S production test, Indole production test Methyl Red test, Voges Proskauer test, and Citrate utilization test.

Table 1 .
Optimization by central composite design CCD model for media composition for biovanillin production

Table 2 .
Optimization by Central Composite Design (CCD) model for media composition

Table 7 .
Bio chemical characterization of MSJM 5 strain

Table 9 .
Effect of ethidium bromide before and afterexpose of MSJM 5 on biovanillin production