A remixed-fermentation technique for the simultaneous bioconversion of corncob C6 and C5 sugars to probiotic Bacillus subtilis

doi:10.21203/rs.3.rs-189021/v1

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A remixed-fermentation technique for the simultaneous bioconversion of corncob C6 and C5 sugars to probiotic Bacillus subtilis

https://doi.org/10.21203/rs.3.rs-189021/v1

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published 30 Mar, 2021

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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 186.1 ± 0.9 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.

General Biochemistry

Biotechnology and Bioengineering

Bacillus subtilis

Corncob

Remixed-fermentation

Biotoxicity

C5 and C6 sugars

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 [2–4]. 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.

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 [7–9]. Among different kinds of agro-forest 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, 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.

2.1 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 348.6 ± 0.6% glucan, 314.2 ± 0.4% xylan and 37.0 ± 0.2% araban. 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.

2.2 Fermentation by Bacillus subtilis

The strain employed was bacillus subtilis CICC 10071. The cells were first cultivated in solid YPD medium. One single colony transferred into the preculture medium (YPD medium) and cultured at 50°C, 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 10g/L corn extract based on dried weight. Xylose, arabinose or glucose was added to the stimulated medium in designed titer.Bacterial fermentation experiments were performed in 250 mL shaking Erlenmeyer flaks 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 2 g centrifugation for 5 min.

2.3 H₂SO₄ acidolysis pretreatment

1000 g corncob powder was mixed with 1% (w/w) H₂SO₄ 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 30min. 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.

2.4 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 solid residue, 5 % (w/v), was mixed with 20 FPIU/g cellulase reagent to produce glucan at pH 4.80. To this, 0.2% (w/v) tetracycline was added to prevent bacterial contamination [18].

2.5 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 250mL conical flask at 50℃ and 150rmp, and PH was adjusted to neutral by 1mol /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 hours and fermented continuously for 24 hours.

Enzymatic fermentation is similar to hydrolysate fermentation. Fermentation was carried out in a 250mL conical flask at 50℃ and 150rmp, and PH was adjusted to neutral by 1mol /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 three hours and fermented continuously for 24 hours.

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.

2.6 Analytical methods

Glucose, xylose, arabinose and inhibitors were analyzed by a high performance liquid chromatograph (HPLC) (Agilent 1260, USA) equipped with an Aminex Bio-Rad HPX-87H column (Bio-Rad Laboratories, USA). The HPLC was operated at 50°C, and 0.005mol/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 was monitored by ultraviolet spectrophotometer (Spectrumlab752s, Leng Optical Technology Co. LTD, Shanghai, China). The turbidimetric method was used as follows: 1 ml fermentation broth was centrifuged in 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 spectrophotometer. 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 OD600nm (y = 1.0000x-0.2006, R2 = 0.999).

3.1 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 cells 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 1.22g/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. This finding may provide a novel solution for the efficient fermentation and bioconversion of inert arabinose present in other carbohydrate rich biomasses. Glucose, xylose and arabinose reached the same maximum cell titer value of around 4.5 g/L and 0.33g cell/ g consumed sugar, despites 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 shaking flask, and it remains to be further studied.

3.2 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 30.68 g/L xylose, the rest 3.01 g/L glucose and 3.68 g/L arabinose. However, no fermentation was detected after 12 hours in the crude xylose broth since all the bacterial cells died. Therefore, we stimulated the fermentation by mixing the three sugars together making it similar to the crude xylose broth. As shown in Fig. 2a, 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 determined and presented the rest of the components other than sugars in Table 1, such as furfural (furfural, 5-hydroxymethyl furfural (HMF)), phenols of vanillin, weak acids of acetic acid and formic acid [19]. These degraded chemicals usually cause intracellular acidification, energy loss and active oxygen accumulation, and inhibit or kill microorganisms [20]. In order to avoid these 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 [21, 22]. Anyway, these added-detoxification operations improves processing-units, cost and environmental problems of industrial production [23]. For overcoming the fermentation inhibition due to bio-toxicity, we tested the basic ‘detoxification method’ by water dilution [24]. B. subtilis yield reached 36.6% by the in-situ and half dilution of crude xylose broth with water (Fig. 2b). 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.

Table 1

Chemical components of hydrolysate and enzymatic hydrolysate
Sample	Chemical components (g/L)
	Glucose	Xylose	Arabinose	Acetic acid	Fomic acid	HMF	Furfural
Hydrolysate	2.35	20.48	3.05	3.42	—	0.12	1.31
Enzymatic hydrolysate	21.92	10.64	—	—	3.11	—	—
Mixed hydrolysate	11.42	15.53	1.76	1.88	1.52	0.06	0.72

3.3 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 works as end-product inhibitors of 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/L 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.

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 221.7 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. 3). B. subtilis effectively utilized xylose just like glucose, but failed to utilize arabinose. For just one day fermentation, 558.3 g/L of C5 and C6 sugars were converted to 186.1 g/L bacterial cells with a yield of 33.3%. Mixed-fermentation technique thus works effectively for the bio-conversion 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. 4 as follows: First, corncob powder was acidolyzed with 1.0% H₂SO₄ with the broth comprising of 18.2 ± 0.2 g glucose, 196.4 ± 0.5 g xylose and 20.2 ± 0.2 g arabinose. Second, the rest solid portion was hydrolyzed to 318.0 ± 3.0 g glucose during enzymatic hydrolysis. Finally, 185.0 ± 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 product’s cost. It is noted that the mixed-fermentation technique dominated the integration process because of the critical bio-toxicity and inhibition on the C5/C6 sugars fermentation by B. subtilis.

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 186.1 ± 0.9 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.

Funding The research was supported by the National Key R&D Program of China (2017YFD0601001), the Key Research and Development Program of Jiangsu (BE2015758) and the National Natural Science Foundation of China (31370573).

Also, the authors gratefully acknowledge financial support from the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Conflicts of interest/Competing interests The authors declare that they have no conflict of interest.

Availability of data and material Not applicable.

Code availability Not applicable.

Authors' contributions XuTong Ma and Yong Xu conceived and designed the study.

Ethics approval Not applicable.

Consent to participate The authors declare that they consent to participate.

Consent for publication The authors declare that they consent for publication.

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Journal Publication

published 30 Mar, 2021

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A remixed-fermentation technique for the simultaneous bioconversion of corncob C6 and C5 sugars to probiotic Bacillus subtilis

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Abstract

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1. Introduction

2. Materials And Methods

3. Results And Discussion

4. Conclusion

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References

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