E ﬀ ect of Substrate Characteristics on the Growth and Sporulation of Two Biocontrol Microorganisms during Solid State Cultivation

: Biocontrol agents are a group of naturally occurring organisms capable of interrupting the lifespan and suppressing the propagation of disease organisms. The use of biocontrol agents o ﬀ ers an environment-friendly and sustainable solution to the synthetic agrochemicals. In this study, we investigated parboiled rice and millets as substrates for spore production of two model biocontrol microorganisms ( Bacillus pumilus and Streptomyces griseus ) under solid state cultivation (SSC) conditions. The e ﬀ ects of cultivation parameters such as initial moisture content, water activity, and cultivation time on microbial growth and spore production were studied. Furthermore, texture proﬁle analysis was performed to test the stress and strain curve and the hardness and stickiness of the substrates. The greatest spore production occurred at 50% moisture content with millets as a substrate, yielding a count of 1.34 × 10 8 spores / g-wet-substrate enumerated with plate count analysis and 1.70 × 10 8 events / g-wet-substrate using ﬂow cytometry analysis. Substrate texture proﬁle was highly correlative to the initial moisture content and substrate type and all proved to be essential process variables in controlling the bacterial growth and sporulation during SSC processes.


Introduction
Soybeans are the foremost oilseed in the United States, recording 90 percent of U.S. oilseed yield.
In the year of 2018, soybeans with~89.2 million planted acres, account for the second-most-planted field crop in the U.S. after corn [1]. Soybean rust is a type of disease that damages soybeans and related legumes. Primarily caused by the pathogenic fungus Phakopsora pachyrhizi [2,3], the disease was first reported in Japan, and has since spread to other parts of Asia, Africa, Australia, and North America [4]. Symptoms first present themselves as small brownish-red dots, or lesions, that appear on the upper side of leaves, with raised pustules forming on the underside [5]. These pustules eventually burst open and release spores, causing the infected leaves to fall off and leading to a premature shutdown of the plant. Consequently, widespread disease can result in severely decreased yield size (up to 80 percent) and smaller seed size, negatively impacting soybean industries [4,5]. While several other methods of management and disease control have been suggested, such as better cultural practices, genetic resistance research, and fungal vaccines, the use of foliar fungicides is still currently the most effective method of managing the disease. Classes such as chlorothalonil and azoxystrobin have been shown to have the most significant impact, but continued research in other fungicides is being conducted [2,6,7].
The broad use of fungicides has led to decreased sensitivity and resistance in P. pachyrhizi [4]. Also, it has been reported that many agrochemicals are toxic and detrimental to both human health

Substrate Preparation
Parboiled long grain white rice and hulled millets (purchased from a local supermarket, Lexington, KY, USA) were used as substrates for B. pumilus and S. griseus. The carbohydrate and protein contents of the two substrates were estimated to be 77.8%, 8.9%, and 71.6%, 10.5% for parboiled rice and millets, respectively [30]. The moisture contents of parboiled rice and millets were analyzed using oven-dry method. In brief, 10 g of parboiled rice and millets were dried in the oven overnight at 105 • C. The initial and final weight were recorded to calculate moisture content. Parboiled rice and millets were mixed with DI water to give three moisture content: 50%, 55%, and 60% (wet basis). Appropriate volume of deionized water was added into 10 g dry substrate to reach the desirable moisture content. The moist parboiled rice and millets were loaded to 250 mL flasks, covered by aluminum foil, and autoclaved for 15 min at 121 • C before inoculation. The raw and autoclaved parboiled rice and millets at different moisture levels were tested for water activities using Aqua Lab water activity meter (Pullman, WA, USA). Triplicate runs were obtained, and data recorded.

Texture Analysis and Microscopic Imaging
Raw and autoclaved parboiled rice and millets at different moisture levels were tested for hardness, stickiness, and stress and strain through texture analysis technique using TA. XT Plus Texture Analyzer (Stable Micro Systems, Surrey, UK) using compression test methods. The condition of the test was at compression speed of 10 mm/sec and return distance of 10 mm. The texture parameters were identified from force-deformation curve, which were hardness (g) Force, stickiness (g), Force, stress (MPa), and stress (%). The elastic modulus, E, was calculated from the slope of the initial linear portion of the stress-strain curves [31].
Scanning electron microscopy (SEM) images of the raw and autoclaved parboiled rice and millet samples were obtained by a FEI Quanta 250 FEG SEM operating at SE mode under low vacuum (0.40-0.65 Torr). Samples were prepared for imaging by freeze-drying using an AdVantage 2.0 bench top lyophilizer (SP Scientific, Warminster, PA, USA). The dried samples (both the surface and crosscut section) were sputter-coated in gold and the imaging was performed at beam accelerating voltages of 2 kV.

Solid-State Fermentation and Spore Production
Subcultures of B. pumilus and S. griseus were inoculated to autoclaved parboiled rice and millets at an inoculation rate of 10 5 colony-forming unit (CFU)/g substrate on dry basis. The cultures were labeled as (BR) B. pumilus inoculation on rice; (BM) B. pumilus inoculation on millet; (SR) S. griseus inoculation on rice; and (SM) S. griseus inoculation on millet, each at three moisture levels. After inoculation, the substrate was stirred with sterilized glass rods. All flasks were then covered with cheesecloth and incubated without shaking at 28 • C for 360 h.
To harvest the spores, culture flasks were transferred into biosafety cabinet and the substrate was mixed with PBS buffer (90 mL). Flasks were then transferred into an orbital shaking incubator and shaken at 200 rpm for 60 min. Subsequently, an aliquot was taken and serially diluted using PBS buffer 10 × stepwise to 10 −7 dilution.

Flow Cytometry Spore Counting
All samples of 10 −4 dilution were evaluated using flow cytometry to estimate the number of spores. In regard to staining, 50 µL of propidium iodide (PI) was used to stain 500 µL of samples. Each sample was incubated in the dark for 5 min at room temperature to permit dye binding. Before injecting to flow cytometer, all samples were homogenized by gentle inversions and vortexed lightly at 5 rpm. Flow cytometry analysis was performed on a BD FASCalibur flow cytometer. PI dyes were excited using 488 nm laser. The flow rate was set up at 550 µL and 2.5 min run time for each sample. All parameters were recorded in logarithmic scale including forwards (FSC) and side scattering (SSC), and up to 1,300,000 total events were counted within G1 gate. The gated spores and cells (R1, R2, R3) events were triggered by using a threshold on both PI and fluorescein isothiocyanate. Dead spores and cells were identified using pulse height for PI fluorescence gated on R3 region. Negative controls were made prior to running actual samples using no PI dyes stained samples. Each sample was recorded in duplicate and averaged for subsequent R3 region event analysis. Flow cytometry results were compared with traditional plate count. Respectively, nutrient agar and tryptone-yeast extract agar were made for sporulated B. pumilus and S. griseus on respective rice and millet. Samples with 10 −4 , 10 −5 , 10 −6 and 10 −7 dilution of BR, BM, SR, and SM with respective moisture levels were plated and incubated for 24h at 28 • C in Heratherm incubator (Thermo Scientific, Waltham, MA, USA). The colonies were enumerated to calculate the number of viable spores.

Statistical Analysis
All experiments were conducted in duplicates or triplicates and the data are presented as means and standard deviations. The statistical analysis, ANOVA and two-ways Tukey's test, was performed by SAS®9.4 (SAS Institute, Cary, NC, US), with a significance level of p < 0.05 for all the data obtained from experiments.

Cultivation Condition Optimization on Agar Plates
Qualitative observations on the microbial growth were made every 24 h over a period of 120 h to determine the suitable culturing temperature and pH for the two strains on agar plates cultured at 25 ± 2 • C and 28 ± 2 • C, each at three pH levels of 5.5, 6.0, and 7.0. The plates stored at 28 • C showed the greatest initial growth in the first three days of incubation. This indicates that over a short incubation period temperature plays a vital role in microbial growth. However, over a week the density of cells on the plates did not show distinct visual differences at the two temperatures. The more prominent variable was the pH of the media. It is clear from Figure S1 that both bacteria preferred neutral pH while did not thrive on media at pH < 6. Since both microbes are natural soil habitants, this observation is reasonable. Although, agar plate optimization will not fully represent the growth condition on real SSC, it provides some essential information on the cultivation parameters of the two strains. Based on that, we further optimized the other cultivation factors such as substrate type and initial moisture content. Figure 1 shows the raw parboiled rice and millet, and the autoclaved parboiled rice and millet at 50% initial moisture content, and B. pumilus and S. griseus grown on parboiled rice and millet, respectively at 50% initial moisture content. It can be seen that the shape, size, and texture of the two substrates in their raw and autoclaved form varied a lot. Such variations can be better defined by characterizing the mechanical and structural properties of the substrates. and initial moisture content. Figure 1 shows the raw parboiled rice and millet, and the autoclaved parboiled rice and millet at 50% initial moisture content, and B. pumilus and S. griseus grown on parboiled rice and millet, respectively at 50% initial moisture content. It can be seen that the shape, size, and texture of the two substrates in their raw and autoclaved form varied a lot. Such variations can be better defined by characterizing the mechanical and structural properties of the substrates.

Texture Profile Analysis (TPA) on Raw and Autoclaved Substrates
A texture profile analysis provides essential information about substrate characteristics in SSC processes. The texture of substrates, parboiled rice and millet, could influence the microbial growth and production of spores. Table 1 shows the Young's modulus of raw and autoclaved parboiled rice and millet with different moisture contents, which were calculated from the stress-strain curves shown in Figure 2. The Young's modulus of raw rice was 421.2 kPa, which was significantly higher than that of raw millet, 118.9 kPa. With moisture incorporated during autoclave process, the Young's modulus reduced remarkably. At 50% of moisture content, the Young's modulus of the rice and millet decreased to 16.8 and 10.1 kPa, which were around 25 and 12 times lower than the raw grains, respectively. As the moisture content increases, the Young's modulus further decreases. At 60% moisture content, the Young's modulus of rice and millet were 4.2 and 2.5 kPa, which were around 400 and 48 times lower than the raw grains, respectively.

Texture Profile Analysis (TPA) on Raw and Autoclaved Substrates
A texture profile analysis provides essential information about substrate characteristics in SSC processes. The texture of substrates, parboiled rice and millet, could influence the microbial growth and production of spores. Table 1 shows the Young's modulus of raw and autoclaved parboiled rice and millet with different moisture contents, which were calculated from the stress-strain curves shown in Figure 2. The Young's modulus of raw rice was 421.2 kPa, which was significantly higher than that of raw millet, 118.9 kPa. With moisture incorporated during autoclave process, the Young's modulus reduced remarkably. At 50% of moisture content, the Young's modulus of the rice and millet decreased to 16.8 and 10.1 kPa, which were around 25 and 12 times lower than the raw grains, respectively. As the moisture content increases, the Young's modulus further decreases. At 60% moisture content, the Young's modulus of rice and millet were 4.2 and 2.5 kPa, which were around 400 and 48 times lower than the raw grains, respectively. It is believed that the texture properties were affected by the ratio of two kinds of starch ylose and amylopectin [32]. Amylose is a linear long chain polysaccharide composed of pr −1,4-linked glucose units, while amylopectin is a highly branched polysaccharide with add glucose units in -1,6 linkage. When the grains were autoclaved in the presence of water, the of both amylose and amylopectin were substantially disrupted. Although after cooling and the cooked grains retrograded, the disrupted amylose and amylopectin chains have rearrang different order, which significantly reduced the stiffness of the raw grains [33]. According t Krishnakumari and Tahumanavan, millet has a total starch content ranged from 64-79% while the total starch content of parboiled rice ranges from 78% to 86% [36]. Based on U FoodData Central, the carbohydrate contents of the two substrates were estimated to be 71.6 77.8% for millets and parboiled rice, respectively [30]. While parboiled rice contains nearly n dietary fiber as compared to ~3.2% in millet, parboiled rice contains much higher level of star bohydrate than that of millet [30]. The higher starch content in parboiled rice could explain t ferent Young's modulus levels of parboiled rice and millet.

Hardness and Stickiness
The hardness and stickiness of parboiled rice and millet were correlated with texture analysis [29]. During cooking or autoclaving, parboiled rice and millet granules absorb wa swell to larger size than its initial state. The particular granule expansion resulted in rupture grain, causing a decrease in the hardness. Moreover It is believed that the texture properties were affected by the ratio of two kinds of starches, amylose and amylopectin [32]. Amylose is a linear long chain polysaccharide composed of primarily α−1,4-linked glucose units, while amylopectin is a highly branched polysaccharide with additional glucose units in α-1,6 linkage. When the grains were autoclaved in the presence of water, the chains of both amylose and amylopectin were substantially disrupted. Although after cooling and drying, the cooked grains retrograded, the disrupted amylose and amylopectin chains have rearranged in a different order, which significantly reduced the stiffness of the raw grains [33]. According to Bora, Krishnakumari and Tahumanavan, millet has a total starch content ranged from 64-79% [34,35]; while the total starch content of parboiled rice ranges from 78% to 86% [36]. Based on USDA's FoodData Central, the carbohydrate contents of the two substrates were estimated to be 71.6% and 77.8% for millets and parboiled rice, respectively [30]. While parboiled rice contains nearly no total dietary fiber as compared to~3.2% in millet, parboiled rice contains much higher level of starch carbohydrate than that of millet [30]. The higher starch content in parboiled rice could explain the different Young's modulus levels of parboiled rice and millet.

Hardness and Stickiness
The hardness and stickiness of parboiled rice and millet were correlated with texture profile analysis [29]. During cooking or autoclaving, parboiled rice and millet granules absorb water and swell to larger size than its initial state. The particular granule expansion resulted in ruptures in the grain, causing a decrease in the hardness. Moreover, according to Cuevas et al., amylose and amylopectin molecules are estimated to leach into the surrounding water above the gelatinization temperature [37]. Those leached amylose and amylopectin molecules are prone to play a part in the stickiness of cooked rice [29,38,39]. Thus, the higher amylose leaching leads to higher stickiness level.
In this study, both parboiled rice and millets were autoclaved at the same temperature and time and compared at identical initial moisture contents (MC): 50%, 55%, and 60%, respectively. Based on Figure 3a, the hardness levels of both parboiled rice and millet decreased as the moisture content increased. The hardness level of parboiled rice significantly decreased from 55% MC to 60% MC. However, the hardness level of millet only showed a gradual/slight decrease from 50% to 60% MC. The less significant decrease of the hardness of millet was probably caused by low extent of amylose leaching. A study by Nuwamanya et al. showed that the extent of amylose leaching of millet, was the lowest amongst different varieties of root, tuber and cereal starches [40].
Fermentation 2019, 5, x FOR PEER REVIEW 7 pectin molecules are estimated to leach into the surrounding water above the gelatinization tem ature [37]. Those leached amylose and amylopectin molecules are prone to play a part in the stick of cooked rice [29,38,39]. Thus, the higher amylose leaching leads to higher stickiness level. In this study, both parboiled rice and millets were autoclaved at the same temperature and and compared at identical initial moisture contents (MC): 50%, 55%, and 60%, respectively. Base Figure 3a, the hardness levels of both parboiled rice and millet decreased as the moisture co increased. The hardness level of parboiled rice significantly decreased from 55% MC to 60% However, the hardness level of millet only showed a gradual/slight decrease from 50% to 60% The less significant decrease of the hardness of millet was probably caused by low extent of am leaching. A study by Nuwamanya et al. showed that the extent of amylose leaching of millet, wa lowest amongst different varieties of root, tuber and cereal starches [40].
As shown in Figure 3b, the stickiness of parboiled rice, from low to high MC, increased d cally from ~45 to 164 (g) force, whereas the stickiness of millet showed gradual decrement from 32 (g) force. It appeared that the hardness of the parboiled rice is negatively correlated with sticki Recent studies described that stickiness is negatively correlated with amylose content and hard [36,41,42]. Stickiness is the susceptibility of the cooked rice adhering to one another, which is the of the rice pulling against the crosshead measured throughout the movement [43]. Neverthele aforementioned, millet has low amylose leaching properties thus exhibited reduction in the stick as MC increases. Generally speaking, parboiled rice and millet demonstrated significant differ in hardness and stickiness at different moisture levels likely due to difference in starch conten composition. Such differences in turn could play a major role in supporting microbial growth.  As shown in Figure 3b, the stickiness of parboiled rice, from low to high MC, increased drastically from~45 to 164 (g) force, whereas the stickiness of millet showed gradual decrement from 76 to 32 (g) force. It appeared that the hardness of the parboiled rice is negatively correlated with stickiness. Recent studies described that stickiness is negatively correlated with amylose content and hardness [36,41,42]. Stickiness is the susceptibility of the cooked rice adhering to one another, which is the force of the rice pulling against the crosshead measured throughout the movement [43]. Nevertheless, as aforementioned, millet has low amylose leaching properties thus exhibited reduction in the stickiness as MC increases. Generally speaking, parboiled rice and millet demonstrated significant differences in hardness and stickiness at different moisture levels likely due to difference in starch content and composition. Such differences in turn could play a major role in supporting microbial growth.

Water Activity
Water activity (a w ) is one of the major factors in the requirements of bacterial growth. Shown in Table 2, raw parboiled rice and millet have the lowest water activity amongst all samples, showing 0.401a w and 0.435a w , respectively. Whereas all moist rice and millet with 50%, 55%, and 60% MC resulted in above 0.90a w with slight increase as moisture content increases. The a w provides an indication of the amount of free water in the substrate and indicates what type of microorganisms could grow on the substrate especially at SSC conditions [44]. Robinson and Nigam suggested that fungal microorganisms are capable of proliferating at lower a w values of 0.6-0.7, making them suitable for SSC at low MC. In general, bacteria grow at higher a w values of about 0.9 [45]; thus all three moisture levels tested in this study, can potentially support the growth of B. pumilus and S. griseus in SSC process. However, the effect of moisture content can be more complicated than just water activity. The effect of initial moisture content on the texture structure of parboiled rice and millet is exhibited by SEM imaging, as shown in Figure 4. The raw grain, both parboiled rice and millet, showed a relatively rough surface, as well as a solid and dense texture, which can be seen in Figure 4a,b. Similar textures were also observed from the images of the crosscut sections of both grains (Figure 4c,d). The autoclaving process led to penetration of moisture and noticeable changes in the microstructure of parboiled rice and millet grains compared to the raw substrates. At 50% moisture content, the surfaces of autoclaved parboiled rice and millet became swelled and smooth (Figure 4e,f). However, significant changes in texture were observed on the crosscut sections. As shown in Figure 4g,h, a large number of pores can be spotted on the crosscut sections of both grains. As the initial moisture content increased from 50% to 60%, the roughness of the grain surface was further reduced (Figure 4i,j), while the pore structure on the crosscut section further developed (Figure 4k,l). Dobraszczyk et al. reported that the porosity of wheat increases in respect of initial moisture content. The increased level of porosity significantly loosens the endosperm structure and contributes to the soft endosperm texture of wheat [46]. The morphology characterization of the parboiled rice and millet suggests that the increased moisture content contributes to an increase in porosity but decrease in hardness of rice and millet after autoclaving, which is consistent with the Dobraszczyk's study. The mechanical and morphological changes of the substrates as a function of initial moisture content and autoclaving could lead to better accessibility of the microorganisms to the nutrients.

Effect of Moisture Content and Substrate Type on Spore Production
Two enumeration methods were used to quantify the viable spores produced after 15-day SSC of the two microorganisms at different moisture contents. By counting the colonies on the agar plate, the highest number of spores were found to be millet with S. griseus subculture (SM), followed by S. griseus on rice (SR), B. pumilus on millet (BM) and B. pumilus on rice (BR) for both 50% and 55% moisture contents. The counted number of spores were significantly lower at 60% moisture. Regardless of moisture contents, it is noted that millet appears as a preferable substrate for spore production. This is possibly due to the fact that millet had lower hardness and stickiness in comparison to parboiled rice, which is more suitable for bacteria to establish and grow on the millet surface. The higher protein content and micronutrient of millet may also contribute to the better microbial growth and sporulation. In general, more spores were produced for S. griseus than for B. pumilus.
Moisture content is a key variable in SSC processes. Over the range of the tested moisture content of 50-60%, the highest spore production, 13.4 × 10 7 S. griseus spores/g-wet-substrate, occurred at 50% moisture on millet (Figure 5a). Note that microbial growth and sporulation depend on several parameters, including the moisture content, hardness and stickiness, and the starch and other constitutions of the substrate. The poor performance at 60% moisture is probably due to the high stickiness that causes substrates to agglomerate, which could have restricted the diffusion of oxygen and nutrient within the substrate. According to Pandey et al., high moisture content could lead to agglomeration of amylopectin masses owing to the surface phenomenon along with the adsorption by grains causing a thick layer of sticky amylopectin film, as a result causing oxygen transfer limitations [47,48]. Consequently, a significant decrease in the production of microbial metabolites is observed. Thus, it is essential to provide and maintain the moisture content at an optimum level. Of course, the optimal moisture level is substate and microorganism dependent. Results from this study suggest that 50% initial MC can provide the best substrate characteristics for parboiled rice and millets that facilitate microbial growth and sporulation of both microorganisms, S. griseus and B. pumilus.
Flow cytometry provides a rapid and facile method to quantify viable cells in a cell suspension. Propidium iodide (PI) is a commonly used membrane impermeant dye that binds to the double stranded DNA; the dye is generally excluded from viable spores so only the dead spores are stained [49][50][51][52]. PI could be selected as the dye exclusion marker [53][54][55] by the intact cell membrane of viable spores, which is a membrane impermeable red fluorescent nucleic acid dyes [56,57]. In this study, cell-impermeant PI dye was used to determine the number of events of respective samples by staining dead spores or spores in which the inner cell membrane had been physically compromised [58][59][60], spores in region 1 (R1) were the dormant or culturable spore sub-population [59,61], and R4 as the inactivated or non-culturable spores. Spores in R2 regions were categorized as the germinated or culturable, but heat sensitive; while spores in R3 represent a population with some damage of an inner membrane, hence promoting PI dyes to gain access to spores ( Figure 6). However, in R3 region, there are other particles that could be detected besides spores such as bacterial cells. Based on Alsharif and Golfrey, R1 represents a live gate around the bacterial population, and R2 and R3 regions correspond to stained bacterial population [62]. Since stained spores could only be detected in R3 region, the number of events in R3 region is calculated and compared to each sample including BR, BM, SR and SM. Based on Figure 5b, the highest number of events is achieved by SM with 50% MC (17.0 × 10 7 particles/g-wet-substrate), followed by 55% MC (12.9 × 10 7 particles/g-wet-substrate), and 60% MC (10.6 × 10 7 particles/g-wet-substrate). While the lowest number of events occurred in BR with 50% MC (2.5 × 10 7 particles/g-wet-substrate). The trend obtained from flow cytometry assay in general matches with the results based on plate count method. The discrepancies could be due to gating errors; however, they could be improved by introducing a secondary dye such as Syto BC, a green fluorescent nucleic acid dye, which stains RNA and DNA in both live and dead spores. The combination of PI and Syto BC could provide a more accurate enumeration on the total spores and the viable spores. Flow cytometry provides a rapid and facile method to quantify viable cells in a cell Propidium iodide (PI) is a commonly used membrane impermeant dye that binds to stranded DNA; the dye is generally excluded from viable spores so only the dead spore [49][50][51][52]. PI could be selected as the dye exclusion marker [53][54][55] by the intact cell membr spores, which is a membrane impermeable red fluorescent nucleic acid dyes [56,57]. In cell-impermeant PI dye was used to determine the number of events of respective sample dead spores or spores in which the inner cell membrane had been physically comprom spores in region 1 (R1) were the dormant or culturable spore sub-population [59,61], an inactivated or non-culturable spores. Spores in R2 regions were categorized as the ge MC (2.5 × 10 7 particles/g-wet-substrate). The trend obtained from flow cytometry assay in ge matches with the results based on plate count method. The discrepancies could be due to gatin rors; however, they could be improved by introducing a secondary dye such as Syto BC, a g fluorescent nucleic acid dye, which stains RNA and DNA in both live and dead spores. The co nation of PI and Syto BC could provide a more accurate enumeration on the total spores and viable spores.

Conclusions
Spores production was achieved by SSC of B. pumilus and S. griseus on parboiled rice and m Substrate type and initial moisture content greatly affected substrate texture characteristics su elasticity, hardness and stickiness, and morphology. The greatest spore production occurred at moisture content with millets as a substrate for S. griseus while lower spore yield was achieved f pumilus also at 50% moisture content with millet. Cultivation parameters such as substrate type nutrient constitution, hardness and stickiness, and water activity all played roles in microbial gr and spore production. Flow cytometry assay gave matching results when compared to plate-c method. Collectively, results from this study lead to a better understanding of the effect of subs characteristics on bacterial growth and sporulation of two biocontrol bacteria during SSC. This s provides important knowledge toward the development of bacterial SSC process for productio biocontrol agents using cheap resources.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/xxx/s1, Figure S1: mizing growth conditions of B. pumilus and S. griseus using agar plates.

Conclusions
Spores production was achieved by SSC of B. pumilus and S. griseus on parboiled rice and millet. Substrate type and initial moisture content greatly affected substrate texture characteristics such as elasticity, hardness and stickiness, and morphology. The greatest spore production occurred at 50% moisture content with millets as a substrate for S. griseus while lower spore yield was achieved for B. pumilus also at 50% moisture content with millet. Cultivation parameters such as substrate type and nutrient constitution, hardness and stickiness, and water activity all played roles in microbial growth and spore production. Flow cytometry assay gave matching results when compared to plate-count method. Collectively, results from this study lead to a better understanding of the effect of substrate characteristics on bacterial growth and sporulation of two biocontrol bacteria during SSC. This study provides important knowledge toward the development of bacterial SSC process for production of biocontrol agents using cheap resources.