Co-culture of Bacillus coagulans and Candida utilis efficiently treats Lactobacillus fermentation wastewater

Co-culture of Bacillus coagulans and Candida utilis was firstly investigated in the efficient treatment of Lactobacillus fermentation wastewater (LFW) containing total organic carbon (TOC) of 22.0 g/L and total nitrogen (TN) of 2.4 g/L. The utilization of lactic acid by C. utilis was responsible for the relief of feedback inhibition to promote the growth of B. coagulans. The removal ratio of TOC by B. coagulans and C. utilis was only 9.1% and 22.7%, respectively, which was improved to 49.0% by co-culture. The removal ratio of TN by B. coagulans and C. utilis was merely 6.3% and 12.5%, respectively, which was also promoted to 44.6% by co-culture. Both the high growth of B. coagulans and the efficient removal of TOC and TN from LFW was achieved with the co-culture, which is not previously reported and very important in the production of probiotics with the resource utilization of LFW.


Introduction
Lactic acid bacteria (LAB) are widely distributed in nature and are of great importance and value in agriculture, animal husbandry, food, medicine and manufacturing industries (Leroy and De Vuyst 2014). LAB in the animal body carry out a variety of physiological functions such as regulating normal gastrointestinal tract flora, maintaining micro-ecological balance, and inhibiting the growth of intestinal pathogenic bacteria, which improves the body immunity (Jahn et al. 1996). Mattia Pia Arena provided a fullest possible overview of the antiviral and antifungal activities ascribed to probiotic LAB (Arena et al. 2018). Zeng et al. (2017) and Mao et al. (2017) used LAB as a safe and convenient oral delivery of bioactive peptide and protein drugs for the treatment of diabetes. Furthermore, LAB is commonly used in the manufacture of yoghurt, cheese, sauerkraut (Zhao et al. 2016) and other fermented food (Di Cagno et al. 2016). With the increasing need for LAB, both their culturing scale and the production of Lactobacillus fermentation wastewater (LFW) are rapidly growing. LFW is generally rich with organic compounds such as sugars and protein, as well as lactic acid produced by LAB, which causes pollution and eutrophication in natural water bodies when it is directly released into the environment. Unfortunately, the treatment technology of LFW, especially recycling it as a culture media for other probiotic strains, has not been reported in the literature up to now.
As a typical bacterial probiotic strain, Bacillus coagulans can produce lactic acid and has an ability in the sporulation to overcome harsh stresses, which can inhibit the growth of intestinal pathogens and regulate the animal intestinal micro-ecological balance (Riazi et al. 2009(Riazi et al. , 2012, and promote animal digestion, and improve animal immunity (Grata and Nabrdalk 2012;Kodali and Sen 2008). Choi et al. (2016) reported that the mixed-culture is a viable approach for the economically feasible production of lactic acid due to the potential use of organic waste for B. coagulans as feedstock.
Candida utilis is another typical eukaryotic probiotic strain that can produce edible protein from various Open Access *Correspondence: haiyan@ustb.edu.cn 1 School of Chemistry and Biological Engineering, University of Science and Technology, Beijing, Beijing 100083, People's Republic of China Full list of author information is available at the end of the article wastes, including bamboo wastewater (Li et al. 2009), rice bran (Rajoka et al. 2004), molasses (Lee and Kim 2001) and potato starch wastewater (Gélinas and Barrett 2007). C. utilis can utilize five-carbon, six-carbon sugars and extracellular organic acids, lactic acid (Oliva and Hang 1979), fumarate, and l-malic acid (Saayman et al. 2000), as carbon sources. In addition, as C. utilis cells are rich in vitamin B and proteins, they can act as a nutrient supplement for livestock, to improve the digestibility of the feed, maintain the microbial balance of the gastrointestinal tract, and enhance the immunity of the animals. Although the individual pure culture of B. coagulans or C. utilis has been well investigated (Yadav and Tarafdar 2012), but no information on the co-culture of them has been reported.
Here the co-culture of B. coagulans and C. utilis using LFW as a medium was investigated, which indicated that the utilization of lactic acid by C. utilis was responsible for the relief of feedback inhibition to promote the growth of B. coagulans. The removal ratios of both total organic carbon (TOC) and total nitrogen (TN) from LFW were all much improved in the presence of B. coagulans and C. utilis simultaneously. The aim in this study is to produce probiotics by the efficient resource utilization of nutrients in LFW, which is not mentioned in the literature and play a vital role in environmental science and biotechnology.

Microbial strains and culture conditions
Bacillus coagulans (ATCC 7050) and C. utilis (ATCC 3052) were bought from Institute of Microbiology, Chinese Academy of Sciences. LFW (Hebei Yiran Biotechnology Co., Ltd.) of Bifidobacterium bifidum was centrifuged (10,000×g for 10 min), filtered (0.22 μ) and sterilized at 121 °C for 20 min, and the initial pH was adjusted to 7.2 by using 40 g/L NaOH solution. B. coagulans grown on beef peptone medium (Chandra et al. 2007) and C. utilis grown on Yeast standard medium (Niu et al. 2011) were used as seeds, inoculated into the pretreated LFW and cultured at 35 °C with the shaking rate of 200 r/min for 4 days. The experiments were carried out in the 100 mL Erlenmeyer flasks with 20 mL culture volume. l.0 mL culture solution of B. coagulans (cell density 4.67 × 10 7 /mL) or C. utilis (cell density 1.53 × 10 7 / mL) was used as inoculum. The samples of three bottles were taken to determine pH, lactic acid, TOC, TN and cell density everyday, respectively.

Identification and count of cell density with flow cytometry
The total biomass of both B. coagulans and C. utilis was measured by a spectrophotometer (722 s, China) at a wavelength of 680 nm (OD680 nm). A flow cytometry (Partec CY-S-3001, German) was further used to identify and count the different cell densities of B. coagulans and C. utilis, respectively. The properties measured include a particle's relative size, relative granularity or internal complexity, and the number of the particle. For flow cytometer measuring, the gain numeric of handle system parameter was set as follows: FSC 155, SSC 290, FL1 248, FL2 361.5. Suspended cells from 0.2 to 150 μ in size was kept by sieving. 10 μL of sample was diluted with 990 μL phosphate buffer solution (PBS; pH 7.0) to 100 fold, and 800 μL diluted sample was prepared in 2 mL transparent test tubes for a flow cytometer. All samples were measured with the excitation and emission wavelengths of 355 nm at room temperature. Each sample was pumped at 2 m/s for about 1.5 min.

Determination of pH, TOC, TN and total phosphorus (TP)
A pH meter (PHS-25, Leici, China) was directly used to measure pH of the culture sample. TOC and TN in culture solution that was centrifuged (10,000×g for 5 min) and filtered (0.22 μm) with the dilution of ultrapure water were determined on a TOC-V CPH (CPH-CPN, Shimadzu, Japan). TP in LFW was also measured by phosphomolybdenum blue spectrophotometry (Li and Liu 2006).

Determination of lactic acid
An amount of 2 mL of culture broth was centrifuged at 12,000×g for 10 min and the supernatant of 200 μL was taken and diluted 20 times with ultrapure water to measure the concentration of lactic acid on a HPLC system (Shimadzu LC-10A TVP, Shimadzu Co., Ltd., Japan) at the wavelength of 210 nm (De Baere et al. 2013). The mobile phase was 99% (v/v) 0.02 mol/L potassium dihydrogen phosphate solution (adjusted pH to 2 with phosphoric acid) and 1% (v/v) acetonitrile using the column of Agilent ZORBAX SB-Aq (150 mm × 4.6 mm, 5 μm) at 35 °C. The flow rate was 1 mL/min and the injection volume was 20 μL.

Main physicochemical indexes in LFW
As shown in Table 1, TOC, TN and TP of LFW in the absence of LAB are 22.1 g/L, 2.4 g/L and 28.5 mg/L, respectively. Furthermore, lower pH 3.8 of LFW may be caused by the production of organic acids such as lactic acid by LAB.
Identification and determination of cell densities of B. coagulans and C. utilis with flow cytometer Figure 1 shows the cell scatter diagram of flow cytometer with FSC as the abscissa and SSC as the ordinate, which indicated that the scatter distribution areas of B. coagulans (R1) and C. utilis (R2) were concentrated on the lower left (Fig. 1a) and upper right (Fig. 1b) of the coordinate system, respectively. The cell scatter diagram in the presence of both B. coagulans (R1) and C. utilis (R2) (Fig. 1c) showed that the cells of B. coagulans and C. utilis could be clearly separated and identified with the different cell size, and their cell numbers could also accurately count with flow cytometer, respectively.
The growth of B. coagulans and C. utilis using LFW as culture medium Figure 2 showed the growth of total biomass (OD 680nm ) of B. coagulans and C. utilis using LFW as a culture medium, which indicated that B. coagulans could slightly grow but C. utilis grew relatively well in pure single culture. The maximum biomass was observed after day 3 in the presence of both B. coagulans and C. utilis, showing that, compared with those of single pure culture, the growth was highly enhanced in co-culture. Further investigation on the growth of two microbial strains in LFW tested by flow cytometer (Fig. 3) showed that the cell densities of B. coagulans and C. utilis were all increased from 6.56 × 10 5 to 19.14 × 10 5 /mL and from 6.56 × 10 5 to 7.26 × 10 5 /mL in the pure culture during the period of 4 days, respectively. However, in co-culture, both the growth of C. utilis (cell density from 6.56 × 10 5 to 8.24 × 10 5 /mL) and especially B.
coagulans (cell density from 6.56 × 10 5 to 215.49 × 10 5 / mL) were much improved at the same time. Figure 4 showed the removal of lactic acid by B. coagulans and C. utilis, indicating that the concentrations of lactic acid was kept almost constant in control and slightly increased in the presence of B. coagulans, respectively, however initial lactic acid of 7.7 g/L was completed removed at 72 h by C. utilis both in single and co-culture, which showed that lactic acid could be produced by B. coagulans but removed by C. utilis, respectively.

Removals of TOC and TN by B. coagulans and C. utilis
As shown in Figs. 5 and 6, both TOC and TN declined slightly in single culture and apparently in co-culture. The removal ratios of TOC by B. coagulans and by C. utilis were 9.1% and 22.7%, respectively, which was improved to 49.0% in co-culture during the period of 96 h. The removal ratios of TN by B. coagulans and by C. utilis were 6.3% and 12.5%, which was also enhanced to 44.6% in co-culture at 96 h. The most efficiencies for the removals of both TOC and TN from LFW were achieved in the presence of B. coagulans and C. utilis simultaneously. Initial pH of 7.0 fluctuated from 6.0 to 9.0 during the period of 96 h (Fig. 7), which have not apparently adversely effects on the growth of B. coagulans and C. utilis.

Discussion
With the increasing need for LAB, both their culturing scale and the production of LFW are rapidly growing, which is generally rich with organic compounds such as sugars and protein, as well as lactic acid produced by LAB (Table 1 and Fig. 4). The release of LFW into natural water body may cause cyanobacterial bloom that can produce toxins such as microcystins and nodularin (Wang et al. 2005), which threatens the safety of drinking  water and human health. Because many nutrients such as sugar, protein and organic acids are remained in LFW (Table 1 and Fig. 4), which may waste the resource if it is simply treated by traditional processing. Unfortunately, the treatment technology of LFW, especially recycling it as a culture media for other probiotic strains, has not been reported in the literature up to now. So we firstly investigated the co-culture of two probiotic strains using LFW as a medium. The size of the cells and their granularity, registered by a flow cytometer, may be used for separation of the mixture (Cruz and Bellakov 1996). Corzo et al. (1999) detected Synechococcus and Prochlorococcus-like populations by flow cytometry in a eutrophic reservoir. Here in the co-culture of B. coagulans and C. utilis in LFW, the cell densities of them could be measured with a flow cytometer (Fig. 1), respectively, which is very important in the identification and determination of the different microbial biomass in co-culture.
Generally, OD 680nm represents the total microbial biomass (Fig. 2), which cannot identify the cell densities of the different microbial strains. Here we successfully identified and measured the cell densities of both B. coagulans and C. utilis by flow cytometer (Fig. 3), respectively. Both B. coagulans and C. utilis are microbial probiotic strains and only pure culture rather than co-culture of them were reported up to now (Niu et al. 2011;Li and Liu 2006). When B, coagulans and C utilis were singly cultured in LFW, C utilis rather than B. coagulans grew well (Figs. 2, 3). However, in the presence of C. utilis, the growth of both especially B. coagulans was much improved (Figs. 2, 3). Firstly, the production of lactic acid may cause the effect of feedback inhibition on the growth of B. coagulans, but the metabolism of lactic acid by C. utilis can relieve the feedback inhibition to promote the growth of B. coagulans (Figs. 3, 4), which is a very important mechanism in co-culture. Oliva and Hang (1979) also found that C. utilis can grow and remove lactic acid from a continuous flow system of pickled wastewater. On another hand C. utilis might provide a number of nutritional factors such as amino acids and vitamins to enhance the growth of B. coagulans. Furthermore, the production of lactic acid from sugar remained in LFW by B. coagulans might also provide carbon source to enhance the growth of C. utilis, so the growths of both were improved each other in co-culture (Figs. 3, 4).
No information is provided on the treatment of LFW containing much amount of TOC and TN (Table 1), here we firstly investigated the resource utilization of LFW as a medium to culture other probiotic strains. The results indicated that the removals of both TOC and TN from LFW in co-culture of B. coagulans and C. utilis were much better than those of single culture (Figs. 5,6). The previous studies also indicated that simultaneous removal of nutrients (ammonium and phosphate) and COD was better by the co-culture of Chlorella vulgaris and Pseudomonas putida than those of single culture, indicating that nutrients uptake capability of C. vulgaris was enhanced in the presence of P. putida.
In summary, the flow cytometer was successfully used to identify and count the different cell densities of both B. coagulans and C. utilis (Fig. 1) using LFW containing much amount of TOC and TN (Table 1) as medium. Compared with B. coagulans, C. utilis relatively grew well in LFW in a single culture (Figs. 2, 3). The growths of two microbial strains especially B. coagulans were much improved in co-culture and the removal of lactic acid by C. utilis was responsible for the relief of feedback inhibition to increase the growth of B. coagulans (Figs. 2,3 and 4). The promotion of microbial growth in co-culture was responsible for the efficient removals of TOC and TN (Figs. 5, 6), respectively, which came true the production of probiotics by the resource utilization of LFW as a medium.

Authors' contributions
JYL and PFS designed and executed the experiments, collected and analyzed all data, wrote the manuscript. HY was responsible for initiation and supervision of the study. HY, QXL and ShA participated in the provided critical review of the manuscript. CHY, XLL, YL and HYZ contributed to experimental theory. QQX provided technical support. All authors read and approved the final manuscript.

Fig. 7
Changes of pH in the culture solution of B. coagulans and C. utilis using LFW as medium