A Remixed-Fermentation Technique for the Simultaneous Bioconversion of Corncob C6 and C5 Sugars to Probiotic Bacillus subtilis

The probiotic strain of Bacillus subtilis presents a promising application potential for the value-added bio-utilization of lignocellulosic carbohydrates. By the combined acidolysis pretreatment and enzymatic hydrolysis, hemicellulose and cellulose constituents of corncob were efficiently converted respectively into fermentable C5 and C6 sugars, mainly including xylose and glucose. B. subtilis grew well in xylose solution while it was hindered completely in the acidolysis broth because of the bio-toxicity of degraded chemicals derived from corncob. A mixed-fermentation technique was therefore developed and performed to blend the acidolysis broth and enzymatic hydrolysis slurry together, by which C5 and C6 sugar molecules were successfully fermented and efficiently utilized for the growth of B. subtilis cells with a yield of 0.33 g cells/g sugar consumed. A net amount of 205.9 ± 9.0 g of B. subtilis powder was obtained from 1000 g of corncob that could improve the economic benefits of the process to around 5–7 times.


Introduction
Bacillus subtilis being the most popular probiotic is an indispensably beneficial microorganism in animal husbandry. B. subtilis can regulate the gastrointestinal micro-ecological balance and enhance the immune performance of animals, thus playing an important role in promoting animals' health and reproduction [1]. Besides being used as a probiotic in fish breeding and poultry raising, B. subtilis is also utilized in water purification, green aquaculture, soil improvement, ecological remediation, etc. [2][3][4]. At present, B. subtilis is mainly produced through the fermentation of starchy materials at an average market price of 55,000 yuan/ton. Therefore, exploring the cheap and non-food sources for the commercial production of B. subtilis probiotics is of greater concern.
A large amount of agro-forest residues are well-known cheap and readily available biomass resources for fermentable C6 and C5 sugars supply, which certainly interests other microbiological fermentation applications besides B. subtilis proliferation [5,6]. Compared with the solo C6 sugar-glucose, present in starchy sources, mannose and galactose are also present in the fermentation broth of agro-forest lignocellulosic residues. Moreover, the C5 sugars-xylose and arabinose are usually inert and hardly fermentable [7][8][9]. Among different kinds of agroforest residual biomass, corncob is a typical xylan-rich lignocellulosic waste and thus the biorefinery technology development of corncob provides a general reference for xylan fermentation [10].
B. subtilis is reported to have potential for glucose and xylose fermentation except for crude lignocellulose derivate sugar broth [11]. The bioconversion of agro-forest lignocellulosic biomass requires at least three pretreatment processing steps: acid pretreatment, enzymatic hydrolysis, and microbial fermentation [12,13]. The initial pretreatment step inevitably releases various complexly degraded chemicals during acidic, hot-water, or steam explosion process, which might be lethal for microorganisms and fermentation [14,15]. In this study, corncob was acidolyzed with dilute sulfuric acid because of its outstanding pretreatment performance and assistance to enzymatic hydrolysis [16,17]. A mixed-fermentation technique was therefore developed to proliferate and harvest B. subtilis from the hydrolyzed crude corncob broth, in which various C6 and C5 sugars were co-fermented readily and effectively to high-value-added bacterial probiotics.

Materials and Strains
Corncob was collected from the local farmer at Jiangsu Province in China, and ground to 20-40-mesh powder. The composition of corncob was determined by the National Renewable Energy Laboratory protocol to be 34.86±0.06% glucan, 31.42±0.04% xylan, and 3.70±0.02% araban [18]. Cellulase reagent (Cellic CTec2, Novozymes) and other chemicals were purchased from Sigma-Aldrich Company Co. Ltd. The strain B. subtilis CICC 10071 was stored on the agar plate at 4°C.

Fermentation by Bacillus subtilis
The strain employed was Bacillus subtilis CICC 10071. The cells were first cultivated in a solid YPD medium. One single colony was transferred into the preculture medium (YPD medium) and cultured at 50°C and 150 rpm for 24 h. The seed cells were cultivated in 250-mL conical flask bottles with 50 mL liquid medium. After 24 h, cells were collected and inoculated into the fermentation media. The initial seed in fermentation media was OD600 = 0.5. The fermentation medium contained 5 g/L yeast extract and 10 g/L corn extract based on dried weight. Xylose, arabinose, or glucose was added to the stimulated medium in a designed titer. The mixed sugar broth contained 20.18 g/L xylose, 3.01 g/L glucose, and 3.22 g/L arabinose. Bacterial fermentation experiments were performed in 250-mL shaking Erlenmeyer flasks containing 50-mL medium, and cultured at 150 r/min and 50°C. The initial bacterial inoculation was at the cell density of OD 0.5, and cell pellet was harvested by 2g centrifugation for 5 min.
H 2 SO 4 Acidolysis Pretreatment 1000 g corncob powder was mixed with 1% (w/w) H 2 SO 4 at a solid-liquid ratio of 1:10 and acidolyzed in a 15-L stainless steel rotary boiler reactor at 150°C and 80 rpm for 30 min. The acidolyzed mixture was cooled down in air and centrifuged. The solid residue was stored in a refrigerator at 4°C for later enzymatic hydrolysis.

Enzymatic Hydrolysis
Solid residue weighing 250 g (ODW) was enzymatically hydrolyzed in a 10-L stainless steel and stirred tank reactor at 50°C and 300 rpm for 48 h. The commercial cellulase reagent (Cellic CTec2, Novozymes) was used to hydrolyze solid residue after acidolysis pretreatment of corncob. The solid residue, 5 % (w/v) solid residue, was mixed with 20 FPIU/g of cellulase activity loading per gram glucan content at pH 4.80 [19].

Hydrolysate Fermentation and Enzymatic Hydrolysis Fermentation
Hydrolysate fermentation is the direct use of undetoxified corncob sulfuric acid hydrolysate for fermentation. Fermentation was carried out in a 250-mL conical flask at 50°C and 150 rmp, and pH was adjusted to neutral by 1 mol /L NaOH. In order to compare the growth of Bacillus subtilis and the utilization rate of sugar in the hydrolysate with different concentrations of sugar, two concentration gradients were set up in this experiment: the diluted hydrolysate and the original hydrolysate. In order to monitor the change of sugar concentration and the growth of bacteria in the hydrolysate in real time, samples were taken every 3 h and fermented continuously for 24 h.
Enzymatic fermentation is similar to hydrolysate fermentation. Fermentation was carried out in a 250-mL conical flask at 50°C and 150 rmp, and pH was adjusted to neutral by 1 mol/L NaOH. In this experiment, two concentration gradients were set up: the diluted enzyme hydrolysate and the original enzyme hydrolysate. Similarly, samples were taken every 3 h and fermented continuously for 24 h.
In order to simplify the experimental steps and improve the utilization rate of sugar, we mixed the hydrolysate and enzymatic hydrolysate in the same proportion for fermentation. Because the sugar concentration of the mixed solution is high, and the mixed solution contains both pentose and hexose, we also set up the mixed solution with two concentration gradients to compare the growth of Bacillus subtilis and the utilization rate of sugar.

Analytical Methods
Glucose, xylose, arabinose, and inhibitors were analyzed by a high-performance liquid chromatography (HPLC) instrument (Agilent 1260, USA) equipped with an Aminex Bio-Rad HPX-87H column (Bio-Rad Laboratories, USA). The HPLC was operated at 50°C, and 0.005 mol/L sulfuric acid was used as the eluent at a flow rate of 0.6 mL/min. The samples were centrifuged, and the supernatant was analyzed by HPLC system. The data was expressed as the mean values and represented in figures. The bars in the figures indicate the ranges of the standard deviation.
The growth and proliferation of B. subtilis were monitored by an ultraviolet spectrophotometer (Spectrumlab752s, Leng Optical Technology Co. LTD, Shanghai, China). The turbidimetric method was used as follows: 1 mL fermentation broth was centrifuged in a 1.5-mL centrifuge tube at 6000 r/min for 5 min, and the supernatant was removed, washed with normal saline, and the mycelium was re-suspended. After dilution with distilled water, the absorbance was determined with a 0.5-cm optical diameter cuvette at the wavelength of 600 nm of spectrophotometry. The obtained absorbance was multiplied by the dilution ratio to obtain the optical density (OD) bacteria. The cell density was converted into cell dry weight by the relationship curve between bacterial dry weight (y) and OD600 nm (y = 1.0000x−0.2006, R 2 = 0.999).

Comparison of Various Monosaccharide Fermentability by Bacillus subtilis
The B. subtilis fermentation of solo C5 sugars (xylose and arabinose) and C6 sugars (glucose) was compared. The experimental results showed that B. subtilis could uptake efficiently the three individual monosaccharides and use them for cell proliferation (Fig. 1). Among the three sugars, the utilization rate of glucose was higher and reached 3.34 g/L/h, while that of two pentose sugars was observed to be relatively slower at 0.82 g/L/h of xylose and 1.22 g/L/h of arabinose.
By comparing individual sugar utilization kinetics, we also found for the first time a very interesting phenomenon that the arabinose utilization rate was somewhat higher than that of xylose. Generally, inert arabinose is difficult to be utilized by microorganisms, but in this experiment, Bacillus subtilis can transform arabinose into probiotic Bacillus subtilis. Therefore, this finding may provide a novel solution for the efficient fermentation and biotransformation of inert arabinose from other carbohydrate-rich biomass. Glucose, xylose, and arabinose reached the same maximum cell titer value of around 4.5 g/L and 0.33 g cell/g consumed sugar, despite the varied kinetics of sugar consumption. When the cell titers increased to 4.5 g/L, sugar consumption was still maintained while no cell proliferation occurred or cell titer remained stable. Moreover, we could hardly detect any fermentation products such as organic acid, acidic acid, or proteins. We speculate the threshold effect for cell titer during fermentation of above-mentioned sugars by B. subtilis in a shaking flask, and it remains to be further studied.

Direct Fermentation of Crude Xylose Broth from Corncob Acidolysis Step
Corncob was pretreated with hot sulfuric acid to produce crude xylose broth, which contained mainly 25.48 g/L xylose, the rest 2.35 g/L glucose, and 3.05 g/L arabinose. However, no fermentation was detected after 12 h in the crude xylose broth since all the bacterial cells died (Fig. 2). Therefore, we stimulated the fermentation by mixing the three sugars together making it similar to the crude xylose broth. As shown in Fig. 3a, B. subtilis consumed almost simultaneously C5 and C6 sugars of xylose for the proliferation of cells. Here, the dominant xylose component presented similar fermentation kinetics as that of glucose, while arabinose showed a weak tendency that was different from individual sugar fermentation and was ascribed logically to the metabolic competition among sugar carbon sources. Compared with the individual sugar fermentation, mixed sugars decreased cell yield to 20.1% because of the mentioned speculated threshold effect of B. subtilis.
For identification of the bio-inhibition factors in the crude xylose broth, we determine and present the rest of the components other than sugars in Table 1, such as furan (furfural, 5hydroxymethyl furfural (HMF)), phenols of vanillin, and weak acids of acetic acid and formic acid [20]. These degraded chemicals usually cause intracellular acidification, energy loss, and active oxygen accumulation, and inhibit or kill microorganisms [21]. In order to avoid these Fig. 2 B. subtilis fermentation of the crude xylose broth negative and inhibitory effects on bacterial fermentation, the detoxification step must be integrated into the biomass-biorefinery process. The common detoxification methods include physical (evaporation, membrane filtration), chemical (resin exchange, alkali detoxification, activated carbon adsorption), and biological (microbial and enzyme) methods [22,23]. Anyway, these added-detoxification operations improve processing units, cost, and environmental problems of industrial production [24]. For overcoming the fermentation inhibition due to bio-toxicity, we tested the basic "detoxification method" by water dilution [4]. In this experiment, we diluted the crude xylose broth with the same volume of distilled water and detected by HPLC that the acetic acid concentration in the diluted crude xylose broth decreased from 3.42 to 1.88 g/L, and the furfural concentration decreased from 1.31 to 0.72 g/L ( Table 1). The fermentation results in Fig. 3b also prove that reducing the inhibitors such as acetic acid and furfural in crude xylose broth can effectively solve the proliferation and fermentation obstacles of B. subtilis using xylose and other hydrolysates. B. subtilis yield reached 36.6% by the in situ and half dilution of crude xylose broth with water (Fig. 3b). Compared with other detoxification methods [25], water dilution always presents overwhelming advantages of being a simple process, being operation ready and low cost for commercial production.

Mixed-Fermentation of Corncob C5 and C6 Sugars to B. subtilis
Solid-liquid separation followed by acidolysis pretreatment is a necessary and effective step before carrying the enzymatic hydrolysis with respect to the inhibitory effects of various degraded chemicals, such as lignin fractions and mono-/oligo-saccharides. The lignin fractions could adsorb and deactivate enzyme-protein components and mono-/oligo-saccharides work as end-product inhibitors of an enzymatic hydrolysis reaction. The separated solids from the pretreatment step were then hydrolyzed effectively to fermentable glucose with 20 FPIU/g of cellulase loading. We obtained 318.4±0.3 g glucose slurry at 88.9±0.2% of enzymatic hydrolysis yield based on the residual cellulose content. This was increased by 27% when compared with the yield of 70% obtained with pretreated materials with no solid-liquid separation step (Fig. 4). Based on the dilution fermentation result, we designed a mixed-fermentation technique for the proliferation of B. subtilis from corncob, by mixing the crude xylose broth with the enzymatic hydrolysis slurry containing 336.6 g/L C6 sugar and 281.7 g/L C5 sugars (xylose + arabinose) and was named as mixed-C5/C6 broth. The mixed-C5/C6 broth bioconversion performed almost similar to the enzymatic hydrolysate during shaking flask fermentation (Fig.  5). B. subtilis effectively utilized xylose just like glucose, but failed to utilize arabinose. For just 1 day of fermentation, 618.3 g/L of C5 and C6 sugars were converted to 205.9 g/L bacterial cells with a yield of 33.3%. Mixed-fermentation technique thus works effectively for the bioconversion of corncob C5 and C6 sugars to probiotic B. subtilis.
Based on a full-processing test of 1000 g corncob, we proposed the integration technique and the mass balance result in Fig. 6 as follows: First, corncob powder was acidolyzed with 1.0% H 2 SO 4 with the broth comprising of 18.2 ± 0.8 g glucose, 256.4 ±7.5 g xylose and 20.2 ± 1.2 g arabinose. Second, the rest solid portion was hydrolyzed to 318.4 ±6.3 g glucose during enzymatic hydrolysis. Finally, 205.9 ± 9.0 g dried cell powder of B. subtilis probiotic was harvested. By a brief techno-economic analysis (TEA), we could improve around 5-7 times the economic income through the bioconversion of corncob to B. subtilis. Here, TEA was based on the average market price of 5000 dollars/t of bacterial probiotics and 100 dollars/t of corncob, as well as the processing cost of around 30% of the product's cost. It is noted that the mixed-fermentation technique dominated the integration process because of the critical biotoxicity and inhibition on the C5/C6 sugars fermentation by B. subtilis.

Conclusion
B. subtilis probiotic cells were proliferated and harvested from corncob by the combined technology of acidolysis pretreatment and enzymatic hydrolysis. To develop a simple and efficient process, C5 and C6 sugars were remixed by blending the acidolysis broth and enzymatic hydrolysis slurry together that caused an effective and in situ dilution of bio-toxic inhibitors during B. subtilis fermentation. We harvested 205.9 ± 9.0 g of B. subtilis powder from 1000 g of corncob and improved the economic benefits around 5-7 times by the integrated bioprocess. This study could provide technical developments for the bioconversion of agro-forest lignocellulosic biomass, especially to high-value-added bacterial probiotics.

Availability of Data and Material Not applicable.
Code Availability Not applicable.
Author Contribution XuTong Ma and Yong Xu conceived and designed the study.