Isolation and Identification of a Bioflocculant-Producing Strain and Application in Rice Vinegar Clarification

: A bioflocculant-producing bacterium S-3# was isolated from fresh sand bones 40-50 cm underground in the desert region of China’s Ningxia Province. It was identified as Paenibacillus sp . based on 16S rDNA sequence along with its biochemical and morphological characteristics. Chemical analysis found that the bioflocculant from S-3# is mainly composed of extracellular polysaccharide. Researchers observed no noticeable change (above 91%) in its flocculating activities in pH range 2.0-9.0 in kaolin suspensions and hypothesized that it could be useful in clarification of rice vinegar (pH 2.9-3.5). Using one-factor-at-a-time experiments, dosage of bioflocculant, temperature of flocculation system and metal cations were investigated for their efficacy on rice vinegar clarification. The Box-Behnken design, an experimental design for Response Surface Methodology (RSM), was also used to create a series of 17 runs to optimize the operating conditions. Quadratic regression models with estimated coefficients were developed to describe the suspended particle removals of rice vinegar. Results showed that the optimal flocculation conditions were 3.94 mg/L bioflocculant, 0.32 mM CaCl 2 and flocculation system temperature of 41.5  C, respectively. These findings were in reasonable agreement with the modeled values. Under these conditions, the increase rate of transmittance (λ = 900 nm) of rice vinegar ranged over 240.63% under these conditions.


Introduction
Flocculants are widely used in industrial processes, from drinking water purification, wastewater treatment, to downstream processes in fermentation processing (Yang et al., 2016;Gassara et al., 2015;Zhang et al., 2020). They can be divided into three categories: Inorganic flocculants, organic flocculants and bioflocculants (Ji et al., 2010;Polasek 2009;Salehizadeh et al., 2018). While the first two categories are widely used due to their effectiveness and low cost. However, these may include the possibility of fatal diseases resulting from extensive usage of alum and the generation of large amounts of sludge, thus introducing new environmental challenges in treatment, regeneration and reuse. In addition, evidence has shown that the second category of polyacrylamide derivatives is a strong carcinogen and has neurotoxic to humans (Semla et al., 2017). Bioflocculants produced by microorganisms have attracted extensive attention due to their nontoxic, biodegradable and benign properties, still more the degradation intermediates will not cause secondary pollution (Salehizadeh et al., 2018;Ajao et al., 2018). Given these considerations, the use of bioflocculants is expected to increase in various fields as a promising substitute for inorganic and chemically synthetic flocculants.
Vinegars have been used as condiment for many years. They are nutritious and have lots of bioactive compounds including amino acids, polyphenols and tetramethyl pyrazine (Ho et al., 2017). These compounds contribute vinegars its unique flavors and also play important roles in preventing and treating human diseases, such as regulating blood lipid levels, weight loss, antifatigue and antitumor (Petsiou et al., 2014;Baba et al., 2013). However, the richness in proteins and polyphenols can induce haze formation in rice vinegar storage, seriously affecting the quality of the product. Currently, the main methods used for vinegar clarification include membrane filtration and flocculation (Lopez et al., 2005;Yin et al., 2019). Among these methods, flocculation is more widely used given its simple process and low cost. Many different types of flocculations have been reported in vinegar clarification, such as bentonite and chitosan (Xiao et al., 2007;Jiang et al., 2013).
Many bacteria have been found to produce bioflocculants and employed in industrial processes (Shahadat et al., 2017). However, maybe due to the limitation of the optimal pH range, there is no previous report on the application of bioflocculants in vinegar clarification (pH 2.9-3.5). Sand bone, made up of sand grains have been found in Ningxia Province, China. The sand bone growing like the roots of tree was 40-60 cm below the ground. It was first reported and named by our research group. In this study, we isolated and identified the novel bioflocculant-producing strain S3# from it and established its application in rice vinegar clarification. The findings from this work are positive and provide useful information for downstream processes in brewing food.

Isolation and Culture of Strain S-3#
Bioflocculant-producing strain S-3# was isolated from fresh sand bones 40-50 cm underground in the desert region of Ningxia Province, China ( Fig. 1 and 2). The enriched medium composition was: 20 g sucrose, 30 g sand (selected form sampling site), 0.5 g MgSO4 ·7H2O, 1.0 g NaNO3, 1.0 g K2HPO4, 0.005 g FeCl2, 20 g agar, 1000 mL distilled water, with a total pH of 7.0-7.5. Using a sterilized needle, the brown material in the hollow part of the fresh sand bones was picked out and then was added into the 250 mL Erlenmeyer flasks containing 50 mL enriched medium and then incubated at 180 rpm for 72 h at 28°C. After that, the sample was spread on the selected agar after a serial of dilution. The composition of the selected agar was: 30 g sucrose, 0.5 g MgSO4 ·7H2O, 2.0 g NaNO3, 1.0 g K2HPO4, 0.005 g FeCl2, 18-20 g agar, 1000 mL distilled water, with a total pH of 7.0-7.5. Strains with high viscosity colony were picked out and inoculated into 250 mL flasks containing 50 mL isolation medium, then incubated at 180 rpm for 48 h at 28°C. 1 mL culture solution was used to measure the flocculating activity.

Identification of Bioflocculant-Producing Strain S-3#
The cell forms and the colony characteristics of strain S-3# on selected agar were observed after 48 h of incubation. The strain was identified by 16S rDNA sequencing. The genomic DNA of S-3# was extracted, then PCR amplification of the 16S rDNA was carried out using universal primers. The PCR conditions were: Denaturation at 98°C for 180 s, 98°C for 25 s with 30 cycles, 55°C for 25 s, 72°C for 60 s and a final extension at 72°C for 600 s. PCR products were purified and sequenced. The synthetic sequence was submitted to the GenBank and analyzed using BLAST. Clustal X 2.1 was used to the alignment of relevant sequences. With the neighbour-joining and maximum-parsimony algorithms using MEGA 5, Phylogenetic trees were constructed (Yang et al., 2015).

Determination of Flocculating Activity and pH Stability
To evaluate the flocculating activity of strain S-3#, kaolin clay suspension was used. 4 g of kaolin clay was dissolved in 1 L distilled water as the Kaolin suspension. 100 mL of this kaolin suspension was added into a 250 mL conical flask which has 1 mL of cell-free supernatant and 3 mL of CaCl2 (1%, w/v). The mixture was shaken for 30 s and then gently poured into a 100 mL cylinder and stand for 5 min. The clarifying solution was collected and measured its OD550. The control group was done similarly, except that the cell-free supernatant was replaced by the production medium. The Flocculating Activity (FA) was calculated as following (Agunbiade et al., 2018): where, A and B are the absorbance of the sample and control group at 550 nm, respectively. To figure out the effect of pH on the flocculating activity, the pH of kaolin clay suspension was adjusted to 2-10. The initial kaolin suspension (pH 7.5) was used as a control group (Pu et al., 2020).

Bioflocculant Purification
To purify the bioflocculant, an equal volume of distilled water was added into the broth and then centrifuged at 14 000 g for 30 min to remove the cells. Triple volumes of ethanol (4C) were added to the supernatant and then the crude bioflocculant precipitate was dried overnight in a desiccator. Then the crude bioflocculant was dissolved in distilled water and 2% (w/v) Cetylpyridinium Chloride (CPC) was added until that the insoluble CPC-bioflocculant complex was no longer formed. The precipitate was re-dissolved in saline solution (0.6 mol/L NaCl), Finally, three volumes of ethanol were added to the supernatant and finally was lyophilized to obtain a purified bioflocculant (Salehizadeh and Shojaosadati, 2002). The total sugar content and protein content of the bioflocculant were determined according to the cited literature (Luo et al., 2005;Liu et al., 2010).

Optimization of Flocculating Conditions
Rice vinegar produced by submerged fermentation was collected and transported to the laboratory using a cooler box containing ice-pack. Transmittance was chosen as the index to measure the flocculating efficiency of the purified bioflocculant in rice vinegar (Xiao et al., 2007). 1 mL the purified bioflocculant solution was added into 100 mL of rice vinegar. The mixture was agitated for 2 min and was allowed to standing for 10 min and then the supernatant was taken for analysis. The transmittance of the supernatant was measured using at 900 nm. The control group was prepared by same procedure except that the distilled water was replaced by the sample solution.
The Increase Rate of Transmittance (IRT) was calculated as follows: where, A initial value; B value after the flocculation treatment.
The bioflocculant dosage, the temperature of the flocculation system and the concentration of cations were the key factors affecting the flocculation efficiency. To get the optimal flocculating conditions of the bioflocculant applied in clarification of rice vinegar, Single factor experiments were used to examine the effects of bioflocculant dosage and various salts and temperature of the flocculation. Based on the above works, these factors were further optimized using a Box-Behnken design. The trials were conducted three times and the average IRT at each run was used as the response variable. The data obtained from RSM was analyzed according to the second-order polynomial equation with the Design-Expert 10.0 software. ANOVA and diagnostics analysis were used to evaluate the quality of analysis model (Wang et al., 2013).
The 16 s rDNA sequences of strain S-3# was determined to be 1458 bp long after the PCR amplification (Fig. 5). Figure 6 was the phylogenetic tree. Based on the morphological characteristics and 16s rDNA sequence analysis, strain S-3# was identified as Paenibacillus sp.

Flocculating Activity and pH Stability Assessment
Flocculating activity of culture broth from the strain S-3# was tested using the Kaolin suspension method, which was 92.8% after 48 h of fermentation. It is much higher than the Klebsiella sp. (86.9%) (Liu et al., 2014) and the Bacillus megaterium (83.8%) (Luo et al., 2016). This suggests that the strain S-3# is able to synthesize bioflocculant effectively. The pH was a key factor that might affect the flocculating activity by changing the ionization degrees of the bioflocculant (Salehizadeh and Yan, 2014). Therefore, different kinds of bioflocculant may have different optimum pH due to their different compositions. The pH stability of S-3# was investigated (Fig. 7). The highest flocculating activity was 95.5% at pH 5.0. The bioflocculant was quite stable with the pH between 2.0 and 9.0, more than 91% flocculation was achieved at this pH range. This result was similar to a polysaccharidebased B40 produced by Bacillus velezensis B40, which maintained a 90% flocculation efficiency at pH 1.0-10.0 (Zaki et al., 2013). However, the optimal pH of most bioflocculants ranged between 5.0 and 8.0, such as Achromobacter sp. (pH 6.0) (Subudhi et al., 2014), Aspergillus flavus sp. (pH 7.0) (Aljuboori et al., 2013) and Halomonas sp. (pH 7.0-8.0) (Shen et al., 2015).

Fig. 3: Colony characteristic of strain S-3#
The high flocculating activity achieved in a wide pH range suggests that this bioflocculant could be applied in acidic neutral and alkalescency conditions. But when the pH was higher than 9.0, its flocculating activity dramatically decreased, this may be due to the bioflocculant having different electric states at different pH (Pan et al., 2009).

Extraction of the Composition of S-3#
About 11.02 g of purified bioflocculant was obtained from 1 L of culture broth. It appeared as a white and hygroscopic powder, with 89.1% (w/w) polysaccharide and 5.8% (w/w) protein. Results revealed that it was a polysaccharide-based bioflocculant and the protein content was very low. To evaluate the effect of polysaccharide and protein on the flocculating activity, the Sevag reagent treatment was used to remove the protein in the bioflocculant and the flocculation activities did not significantly declined. This confirmed that extracellular polysaccharides were the key effective component for the flocculation ability.

Factors Affecting the Bioflocculant on Rice Vinegar Clarification
In this study, we found that S-3# possesses high flocculating activity in kaolin suspensions and the flocculating activity has no remarkable change at pH from 2.0 to 9.0. Therefore, it could be used in rice vinegar clarification (pH 2.9-3.5).
The effect of bioflocculant dosage (Fig. 8) showed an over 48% increase rate of transmittance in the range of 1-11 mg/L, with the maximum IRT of 133.62% observed at optimum bioflocculant dosage of 3.0mg/L. Figure 8 also showed that the dosage affected the flocculation efficiency. When bioflocculant is insufficient, the bridging phenomena cannot efficiently formed. In contrast, an abundance of negatively charged bioflocculant can cause the repulsion of negatively charged particles and similarly reducing effective volume leading to poorer stability. This is consistent with the flocculation characteristics of polysaccharide flocculants produced by other microorganisms (Gong et al., 2008;Kwon et al., 1996). The effect of the temperature of flocculation system was also investigated (Fig. 9). Flocculating activity increased with temperature increase up to the maximum value of 40℃, thereafter slowly decreased. This could be due to the fact that the high temperature changed the spatial structure of the bioflocculant and induced the lower flocculation efficiency.
To achieve high flocculating activity, metal cations are often added as they may enhance the bridging and neutralization of bioflocculants in the process of flocculation (Sobeck and Higgins, 2002;Kim et al., 2013;Vimala et al., 2020). Figure 10 shows the effect of cations on flocculation. Apparently, trivalent ions (Al 3+ and Fe 3+ ) and bivalent ions (Ca 2+ , Mg 2+ and Fe 2+ ) were more effective than monovalent ions (Na + and K + ). Similarly, the bioflocculant S-14 produced by Serratiaficaria sp. was effective with metal ions (Ca 2+ and Mg 2+ ) (Gong et al., 2008).
Given high cost and other considerations (Fe 2+ is easy to oxidize and bring color into the system, Al 3+ is easy to lead to Alzheimer's disease and other disadvantages), Ca 2+ appears to be the ideal coagulant aid. In Fig. 11, the high flocculation efficiency was achieved when Ca 2+ concentration was 0.3 mM.

Using RSM for Optimization
Bioflocculant dosage, flocculation system temperature and CaCl2 were significant factors influencing flocculation. The following experiments using three-level, three-factor Box-Behnken design model with 17 experiments were performed to optimize these factors, the average of both increase rate of transmittance at each run was used as the response variable (Table 1), The statistical testing was carried out with the Fisher's statistical method for Analysis Of Variance (ANOVA) ( Table 2). The actual response values from the ANOVA analysis were compared with predicated response values, showing a statistically valid low probability value of P (Pmodel = 0.0009) indicating that the equation is very reliable in predicting the flocculating efficiency. The lack of fit value was insignificant (p = 0.1292), indicating that the equation was adequate for predicting flocculating efficiency. The adequacy of the model was indicated by the determination coefficient (R 2 = 0.9504), which accounted for 95.04% of the response variability.   These surface plots, therefore, allow for visualization of the optimum levels of each variable to maximize the flocculating efficiency. The optimal conditions for the bioflocculant were obtained using the "numerical optimization" function within the Design-Expert 10.0. With 3.94 mg/L bioflocculant, 0.32 mM CaCl2 and flocculation system temperature of 41.5C, the highest increase rate of transmittance was predicted to be 241.04%. To verify the reproducibility of the model, all experiments were performed under the optimal conditions. The results showed that the increase rate of transmittance was 240.63, 242.51 and 243.64%. This suggested that the model can reasonably optimize the flocculation conditions and predict the flocculation efficiency for rice vinegar clarification.

Conclusion
The bacterium S-3#, isolated from the fresh sand bones, was identified as Paenibacillus sp. It has shown to be able to synthesize bioflocculants effectively. Moreover, the bioflocculant possessed wide range of adaptations at pH 2.0-9.0. It was found that extracellular polysaccharide was the main constituent of the bioflocculant. The Box-Behnken design showed that the optimal flocculation conditions used in rice vinegar clarification were 3.94 mg/L bioflocculant, 0.32 mM CaCl2 and flocculation system temperature of 41.5C, respectively. The increase rate of transmittance was over 240.63% under these conditions. Suggesting that the bioflocculant may have promising applications in clarification of brewing food items.
Although bioflocculant has many advantages over other flocculants, the bioflocculant is much expensive due to the high cost of the productive process. To overcome this impediment, future emphasis should be placed on finding out a low-cost medium. The application of bioflocculant in the production of beer will also be studied in the future.