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, OD680nm 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.