2.1. Microorganism

Bacterial strain B. subtilis PW 12 (MTCC 10402) was procured from CSIR- Microbial Type Culture Collection Centre (MTCC), Chandigarh, Punjab, India. The culture was preserved in 40% glycerol and kept at −20°C. The bacterial culture was maintained on nutrient agar slants and plates for routine work. The slants were kept at 4°C and periodically subcultured in nutrient broth medium.

2.2. Preparation of Seed Culture

Pure culture was used to inoculate 100 mL of Tryptic Soy Broth medium contained in a 250 mL Erlenmeyer’s flask and cultured in an orbital shaker at 37°C and 150 rpm for 24 h.

2.3. Medium Components and Culture Conditions

Medium components such as glucose, peptone, yeast extract, soya peptone, NaCl, MgSO..7H.O, and KH.PO. were chosen, while culture conditions like temperature (°C), pH, agitation (rpm) and inoculum size (% v/v) were selected for the study. The incubation period for the strain was chosen as 48 h following a trial.

2.4. Statistical Experimental Design for Optimization

2.5. Evaluation of Biomass Production

Biomass production of B. subtilis PW 12 grown in optimized medium was evaluated in a laboratory bioreactor. The dry weight of the bacterial cell biomass was determined by centrifuging 10 mL of culture sample at 5000 g for 20 min, drying at 80°C overnight, and weighing the resulting dry cell biomass in grams per liter.

3.1. Screening of Significant Nutrient Components Using Plackett-Burman Design

The Plackett-Burman design is a key tool in screening the effects of the variables on the final response [18]. The biomass yield showed variation across the 15 experimental runs (Table 3). ANOVA results for PBD revealed that only 4 out of 11 variables had a significant effect on the response, i.e., biomass yield. Soya peptone, glucose, pH, and inoculum size had a significant influence on biomass production (p < 0.05) [Table 4, Figure 1]. Hence, only these factors were used for further optimization experiments. The polynomial equation for biomass yield is represented by Equation 3.

Table 3 : Design and responses of the Plackett - Burman Design (PBD).

Table 4: Analysis of variance for the experimental results of the Plackett - Burman Design (PBD).

Figure 1: Pareto chart showing standard effects of 11 variables on biomass production of B. subtilis PW 12. [Click here to view]

The linear regression coefficient of determination, R², was 0.99, which indicated that the predicted model could account for 99.17% of the variability in the experimental data. The model was fit to the data, as the lack of fit was found to be insignificant (p > 0.05). Since the curvature was significant (p < 0.05), a higher order model was necessary to optimize the levels of significant variables.

The Selection of medium components and their concentrations is fundamental to medium optimization, as the availability of substrates influences growth rate, and the limitations of different substrates can significantly affect the nutritional value of microbial biomass [19]. An adequate supply of carbon and nitrogen, together with ideal growing conditions, are the most essential requirements for bacterial growth. The variables investigated in the present study were selected to account for the sources of carbon (glucose), nitrogen (peptone, yeast extract, and soya peptone), inorganic nutrients for the supply of phosphate, sulfate, and minerals (KH₂PO₄, MgSO₄.7H₂O, NaCl) and culture conditions (temperature, pH, agitation, and inoculum size). Since no other optimization experiments had been conducted on B. subtilis PW12, the medium components and culture conditions for the study were chosen based on prior research on other strains of B. subtilis. Prior to screening, OFAT studies provided information on the ranges of concentrations for each individual component that should be used in the Plackett-Burman design.

Screening experiments using PBD showed that soya peptone, glucose, pH, and inoculum size had substantial impacts on biomass yield. The nitrogen source has a key role in determining the rate of bacterial growth [20]. Soya peptone had the highest effect on biomass production by B. subtilis PW12. Other nitrogen sources used in the PBD study, such as yeast extract and peptone, had an insignificant effect on biomass production. The effective use of soya peptone or soya protein hydrolysate as nitrogen sources by some strains of B. subtilis has been reported, where high cell density biomass production was a prerequisite for the synthesis of certain secondary metabolites and industrially important enzymes [21-23]. Several prior investigations, in contrast to the current study, have utilized other organic nitrogen sources, such as peptone and yeast extract, to produce biomass using some specific strains of B. subtilis [24-26]. In the present study, the enhanced production of biomass may be attributed to the complex mixture of amino acids, short peptides, carbohydrates, etc. contained in soya peptone. Despite being a non-essential amino acid, glutamate is the one amino acid that B. subtilis prefers [27,28]. According to the study by Leibs et al. [29] the exponential growth phase of B. subtilis showed considerable intake of glutamate, aspartate, serine, and alanine from the growth media. Moreover, soya peptone has a high reserve of amino acids and a total glutamic acid concentration higher than other amino acid contents [30]. Together with other growth factors, the presence of these amino acids might have helped the bacteria produce more biomass. Additionally, it is possible that B. subtilis may efficiently use soya peptone as it can produce the enzyme protein glutaminase (PG), which has the ability to hydrolyze amides of glutamine in proteins and thereby enhance the solubility of plant proteins. During fermentation, B. subtilis may produce extracellular proteases that can self-activate protein glutaminase, which is released in the inactive pro-enzyme form and can facilitate efficient substrate utilization by the bacterium [31].

The effect of glucose was also statistically significant on the biomass production of the bacteria. Previous studies also show that glucose is a preferred carbon source for B. subtilis and other Bacilli [32-34]. The phosphoenolpyruvate: sugar phosphotransferase system (PTS) in B. subtilis aids them to take up and phosphorylate the sugar, and the presence of PTS dependent and independent glucose transporters also facilitates the bacteria in efficiently utilizing glucose as the carbon source [32,35]. Additionally, as glucose is a readily metabolized carbon source, it may accelerate the fermentation process and enhance the production of bacterial biomass. Previous studies have found that glucose was the preferred carbon source for obtaining a high biomass yield in B. subtilis strains for the synthesis of several secondary metabolites and enzymes that are linked to high cell densities using batch or fed batch fermentation methods [21,36,37].

Among the culture conditions, pH and inoculum size significantly affected biomass production. Since pH is the primary control of bacterial metabolism, optimum pH is necessary for their maximum biomass production. Besides pH, inoculum size also showed a significant impact on biomass production. As inoculum concentration determines the lag phase of bacterial growth, the addition of adequate inoculum of bacteria at the appropriate developmental stage is important to achieve increased biomass yield [38].

In the present study, temperature and agitation had the least effect on biomass yield among the culture conditions. Previous studies on B. subtilis strains suggest that their optimal temperature and agitation ranges are 30°C-37°C and 200–250 rpm, respectively [34,39,40]. The temperature and agitation ranges applied in the present PBD study were ideal for B. subtilis PW12 growth. Consequently, the temperature and agitation values in the CCD optimization study were set at 37°C and 200 rpm, respectively.

3.2. Optimization of Biomass Production by RSM and CCD

The Central Composite Design was carried out to optimize the levels of significant variables to maximize the biomass yield. The design matrix and the associated experimental results show that biomass yield varied across the experimental runs (Table 5). The second-order polynomial equation for biomass yield is represented by the following equation [Equation 4].

Table 5 : Design and responses of the central composite design (CCD).

The coefficient of determination, R², which was 0.98, indicated that 98.71% of the variability in the response could be elucidated by the model (Table 6). ANOVA was conducted to assess the statistical significance of the polynomial equation, and the results showed that the regression was statistically significant (p < 0.05). The significance of the regression coefficient of the model reveals that the linear effect of all variables except pH (p > 0.05) on the maximum biomass production was significant (p < 0.05) (Table 6). However, the squared effects of all the variables were significant (P < 0.05) and except for the soya peptone - pH interaction (P > 0,05), all other interactions were statistically significant (P < 0.05). The lack of fit of the model was insignificant (P > 0.05) indicating that the model fits the data. The significant interactions of variables described by the model are shown in contour plots and three-dimensional response surface plots [Figure 2].

Table 6 : Analysis of variance for the experimental results of the Central Composite Design (CCD).

Figure 2: Counter (left) and response surface (right) plots representing significant interaction of variables on biomass yield: (A) Glucose vs. soya peptone, (B) Inoculum size vs. Glucose, (C) Inoculum size vs. soya peptone, (D) pH vs. glucose, and (E) Inoculum size vs. pH. [Click here to view]

Carbon and nitrogen sources are absolutely essential for bacterial growth. In the present study, the interactive effects of glucose and soya peptone also significantly contributed to biomass production. The response surface and contour plots show that the optimal concentrations of glucose and soya peptone were around 14 g/L and 27 g/L, respectively [Figure 2A]. In concordance with the current investigation, a single factor optimization study by Naveed et al. [36] revealed that the addition of 1.5% glucose and 2.5 % soya peptone in the fermentation medium of a newly isolated strain, B. subtilis BSN314, caused enhanced biomass production in the bacterium. However, studies by Zhong et al. [21] showed that an optimized medium containing 30.70 g/L of glucose and 2.4 g/L of total nitrogen from soya bean meal hydrolysate was beneficial for high cell density cultivation of B. subtilis ZK8 in fed batch fermentation. Investigation of Yue et al. [41] demonstrated that cell concentration of B. subtilis ZK-H2 could be increased to 7x10⁸ cfu/mL in an optimized medium containing glucose and soya peptone, where the optimal concentration for glucose and total nitrogen concentration from soybean protein were 21 g/L and 4.0 g/L, respectively. Zhong et al. [42] used soya bean meal hydrolysate and glucose in the medium of a wild type strain, Bacillus sp. H-18W, for the production of chiral acetoin, a metabolite associated with fast cell growth. Nguyen [22] used glucose at 5.62 g/L and soya bean peptone at 13 g/L in the optimized medium for higher biomass production by B. subtilis Natto, whereas a study by Ullah et al. [43] showed that soya peptone and glucose at 1% w/v in the medium resulted in improved cell growth of a probiotic strain of B. subtilis and in contrast to the present study, higher concentrations of soya peptone caused a decline in cell growth. Stamenkovic et al. [33] reported that biomass production of B.subtilis NCIM2063 could be enhanced when the growth medium was supplemented with 10 g/L of glucose. Study by Cho et al. [25] demonstrated that supplementation of the initial medium with 10 g/L glucose and 50 g/L peptone in a fed batch culture caused an increase in the cell density of B. subtilis however, exhaustion of glucose after 6 h was also reported. Since B. subtilis can work on soya-based proteins, Wang et al. [44] included soy bean curd as well as soya peptone at concentrations of 12.2% w/v and 5.7% w/v respectively, in the medium for metabolite production and growth of B. subtilis. Study by Yanez et al. [45] reported that even defatted soy flour (40 g/L) could be used in the growth and metabolite production medium of B. subtilis CtpxS2-1 and was found to improve bacterial growth. From the present study, it can be concluded that B.subtilis PW 12 strain could also successfully utilize glucose at less than 15 g/L and soya peptone at less than 30 g/L in the culture medium for attaining maximum growth.

Soya peptone and glucose had significant interactions with inoculum size as well [Figures 2B,]. In the current investigation, it was shown that biomass production rose in tandem with inoculum size. A high inoculum concentration can promote growth up to a certain point, but afterwards, it causes the microbial activity to decline because of the depletion of nutrients in the medium. Conversely, a low inoculum concentration decreases the cell concentration, which may affect the amount of bioproduct produced. At lower cell concentrations, it takes a long time to attain optimum growth [46]. The optimum inoculum size in the current investigation was 11.03% v/v, indicating that the soya peptone and glucose-based optimized medium had sufficient nutrients to maintain a higher inoculum concentration to enhance the growth of the bacterium. Study by Huang et al. [47] on B. subtilis biomass production shows that 8% v/v of inoculum size may be ideal for achieving optimal production.

Though the linear effect of pH was not significant, its interaction with glucose and inoculum size was significant [Figures 2D, E]. Response surface and contour plots show that maximum production of biomass was obtained at pH 7. The optimal pH for B. subtilis strains varies depending on the specific strain and the conditions of the study. In general, the optimal pH for B. subtilis strains appears to range from pH 4.0 to pH 8.0, depending on the specific strain and the activity being measured, and previous studies show that most of the B. subtilis strains prefer pH 7 for their optimal growth [22,26,36]. Acidic pH may result in rigid and ordered membranes, which can affect the membrane dynamics of the bacterium [48].On the other hand, alkaline conditions may cause membrane disorder [49] and hence maintaining optimal pH is crucial for the membrane properties and thereby the growth of B. subtilis. At the optimum pH, glucose at a concentration of 14.07 g/L contributed to the maximum biomass production [Figure 2D]. pH variation during bacterial growth is greatly influenced by the medium composition, particularly the carbon source. Since glucose is a reduced carbon source, more energy may be derived from its oxidation, leading to greater bacterial proliferative potential, and there might be an increased proton flux causing acidification of the medium during the exponential phase of bacterial growth [50]. Therefore, maintenance of optimal pH throughout fermentation is desirable. At the optimal pH, an inoculum size of 11% v/v was significant for enhanced biomass production [Figure 2E].

By using the equation, the optimal concentrations for the variables were predicted. Concentrations of soya peptone (a) glucose, (b) pH (c) and inoculum size (d) obtained from the maximum point of the model were 27.56 g/L for A 14.07 g/L for B, 7.09 for C and 11.03% v/v for D respectively. The model predicted a maximum biomass yield of 14.19 g/L for this point.

In the present investigation, inorganic nutrients, including NaCl, MgSO₄ .7H₂O, and KH₂PO₄ had no significant effect on biomass production. While this does not necessarily negate their importance for bacterial growth, the findings of this study suggest that among the media components, soya peptone and glucose notably influence biomass production. Inorganic nutrients are the most common inclusions in bacterial growth mediums as bacteria require anions like phosphates and sulfates, and cations like sodium, potassium, magnesium, iron, and calcium. These compounds are essential for the synthesis of nucleic acids, proteins, and essential cofactors. The quantity of inorganic salts that must be added to the fermentation medium varies depending on the nutritional requirements of the organism, the composition of the culture medium, and the type of desired end product [51]. In the present study, inorganic nutrients were found to have an insignificant impact on bacterial biomass production. Since this study did not center on the production of specific primary or secondary metabolites by the bacteria, there might be room for flexibility in the required quantities of these inorganic salts, thus making their optimal concentration less strict in the bacterial growth medium. The bacteria were able to proliferate at both low and high levels of the inorganic nutrients applied in the PBD. As a result, in the final optimized medium, the concentrations of these inorganic nutrients were set at the lowest level utilized in PBD as 5 g/L, 2 g/L, and 2 g/L for NaCl, MgSO₄, 7.H₂O, and KH₂PO₄, respectively (Table 1). Furthermore, soya peptone, the nitrogen source in the medium, usually contains an ash content of less than 15% which indicates the presence of minerals like calcium, magnesium, and potassium. However, soya peptone is reported to have a very low inorganic phosphorous content [52].The essential minerals and vitamins present in the nitrogen sources might have been utilized by the bacteria for growth.

3.3. Validation of the Optimized Culture Medium

To validate the modeling results, triplicate experiments were done using the optimized conditions. The predicted maximum yield was 14.19 g/L, and the average value obtained in the experiments was 14.29 ± 0.23 g/L, which was in agreement with the predicted yield of the current optimization study. The biomass yield obtained in this investigation was similar to that reported in the study of Ma et al. [23] in which a fed batch fermentation experiment with a mutant strain of B. subtilis 168 mut-16# was conducted. Feeding of soya peptone and hydrolyzed starch to the medium increased the dry cell weight of the bacteria, and the highest dry cell weight obtained was 14.3 g/L which was comparable to the findings of the present study. However, a statistical optimization study conducted by Zhong et al. [21] on B. subtilis ZK8 reported 77.5 g/L dry cell weight of the bacterium in fed batch culture and 20.38 g/L in batch culture. Yadav et al. [53] used peptone as the nitrogen source in the growth medium of B. subtilis and a maximum dry cell weight of 7.5 g/L was obtained after optimization. Koim-Puchowska et al. [27] used yeast extract (4 g/L) and soluble starch (40 g/L) as nitrogen and carbon sources, respectively, for the growth of B. subtilis natto BS19 and resulted in a biomass yield of less than 6mg/mL. Studies of Nguyen [22], Stamenkovic et al. [33], Naveed et al. [36], and Ghasemi and Ahmadzadeh [54] reported biomass yields as 3.033 g/L, 6 g/L, 3.81 g/L, and 0.5 g/L, respectively, for different B. subtilis strains.

The biomass yield achieved in this study emphasizes the importance of medium optimization in augmenting microbial biomass production. It also suggests the potential scalability of this medium to an industrial level for the production of single-cell protein from B. subtilis PW12. Considering the former in regard to manufacturing costs, finding alternatives to the main nutrient of the optimized medium i.e., soya peptone, could prove effective in enhancing the production efficiency of the scale-up process. However, cost issues associated with this nitrogen source have been addressed in some prior research, and cost-effective methods of soya peptone synthesis using enzymatic hydrolysis of soya beans have been proposed [55,56]. Substrates based on soya protein, such as defatted soy flour and soy bean curd, have been used in experiments to produce biomass and metabolites by fermentation with B. subtilis [44,45]. Considering the adeptness of B. subtilis PW 12 at utilizing soya peptone, delving deeper into the effects of cost-effective nitrogen sources extracted from soybean meal on bacterial growth could offer valuable insights.

Moreover, the most critical juncture of an entire scale-up process is the transition from shake flask to laboratory bioreactor. It is imperative to understand the hydrodynamic behavior of the bioreactor, including mass and heat transfer, mixing, and aeration, especially in aerobic fermentation, to verify the efficacy of the scale-up process. [34] This information is pertinent to the current study as well, so as to achieve yields in the bioreactor that are equivalent to or surpass those in shake flasks under the same conditions.

The goal of the current study was to optimize the biomass yield as well as the nutritional quality of a beneficial probiotic bacterium, B. subtilis PW 12 that could be used as a single cell protein or protein supplement in aquafeeds. According to Sakarika et al., the nitrogen content of the culture medium may be positively correlated with the nutritional quality, especially the protein content of the microbial biomass [19]. Since the optimized medium is rich in nutrients, the bacteria may synthesize and accumulate more protein due to their ability to proliferate extensively, resulting in a high cell density. Consequently, the biomass can be utilized as microbial protein in aquafeeds. The current investigation validates that B. subtilis grows optimally in a medium based on soya peptone, and glucose, which are the standard ingredients of microbiological media. Since the medium and culture conditions have been optimized to enhance the growth and nutritional quality of the test bacterium, the growth of other strains of B. subtilis, particularly those that can achieve high cell densities and other probiotic strains, can also be evaluated in this optimized medium for increased biomass production. This study has only taken into account the maximal biomass yield; nevertheless, due to the fact that bacterial cells are dynamic, they may produce extracellular and intracellular enzymes, proteins, and metabolites when stimulated by external factors, including medium components or the culture conditions. However, as the study has not examined the production of any particular antimicrobial compound, metabolite, or enzyme by B. subtilis PW12 in the optimized medium, future investigations on these facets may assist in evaluating the transferability of the medium to other similar B. subtilis strains. Given the under-researched status of B. subtilis PW12, thorough exploration is needed for a comprehensive grasp of its metabolic, antibacterial, probiotic, and nutritional properties to probe its potential application in aquaculture and related fields.