This study provides clearly demonstrated the growth inhibition of the harmful cyanobacterium C. raciborskii and adsorption of CYN toxin adsorption by the yeast A. pullulans. Co-cultivation of C. raciborskii with yeast cells showed decline in C. raciborskii cell density, with total cell lysis within 4 days. This is the first study on the anti-cyanobacterial activity of fungi against C. raciborskii. Nevertheless, KKUY0701 strain was found to remove all M. areuginosa cells within 72 hours in batch experiment (Mohamed et al. 2020). Additionally, the removal efficiency of C. raciborskii cells by KKUY0701 strain can be compared to those of other anti-cyanobacterial fungi such as Trichoderma abietinum, Trichoderma citrinoviride and Lopharia spadicea, which eliminated all M. aeruginosa cells within 48h (Jia et al. 2010b; Mohamed et al. 2014). Our study also showed that the filtrate of A. pullulans could suppress C. raciborskii growth, indicating involvement of anti-cyanobacterial substances excreted by this yeast into the medium. Thus, our results support the finding of other studies stating that Trichoderma citrinoviride and A. pullulans inhibited cyanobacterial growth through the extracellular excretion of anti-cyanobacterial substances into the medium in lieu of direct attack (Mohamed et al. 2014; 2020).
In this study, A. pullulans growth increased after one day of co-incubation with C. raciborskii cells and became greater than in control cultures (i.e., YMA medium). The ability of A. pullulans to grow in medium without carbon source suggests that it could decompose and utilize cell constituents of C. raciborskii for its growth. These results support the finding of previous studies that some yeast strains e.g., Saccharomyces cerevisiae and A. pullulans lysed cyanobacterial cells and used their contents as a carbon source (Möllers et al. 2014; Mohamed et al. 2020).
The anti-cyanobacterial activity of A. pullulans towards C. raciborskii may be attributed to the production for lytic enzymes (e.g., N-β-acetylglucosaminidase), which can digest peptidoglycan, the main constituent of Cylindrospermopsis cell wall (Di Francesco et al. 2015). However, such inhibitory and lytic compounds produced by A. pullulans should be further isolated and characterized.
In addition to growth inhibition of C. raciborskii, A. pullulans removed the released CYN toxin in the culture medium. This was manifested by the decline in CYN concentrations in the medium of yeast- treated cultures but without significant change in filtrate-treated cultures. This observation indicates that CYN toxin could be eliminated through biodegradation by living yeast cells or adsorption onto yeast surfaces. Our adsorption experiment revealed a reduction in CYN concentrations in the presence of heat-inactivated and living yeast. However, inactivated yeast caused greater decrement in CYN concentrations than living yeast. This suggests that the metabolic and enzymatic activities were not involved in the reduction of CYN concentrations (i.e., not biodegradation), but the decline in toxin concentrations was rather due to its binding to yeast cell walls. Concomitantly, other studies demonstrated the adsorption of mycotoxins (Luo et al. 2015) and microcystins (Mohamed et al. 2020) by yeast strains. Our results also showed that Freundlich isotherm parameters including K (i.e., adsorption capacity) and 1/n (i.e., the adsorbent affinity) are correlated, and therefore, the adsorption of CYN toxin by KKUY0701 strain is suitable. In our study, the data of isotherm parameters (K = 3.3, and 1/n = 0.7), can be compared with those (K = 2.9, 1/n = 0. 84, respectively) recorded in the literature for CYN adsorption by activated carbon (Liu 2017). The adsorption capacity of KKUY0701 yeast strain for CYN toxin increased with the increase of initial CYN concentrations, up to a certain concentration (40–50 µg L− 1) and then decreased at higher concentrations (60–100 µg L− 1). This reflects that CYN adsorption this yeast strain is a process reaching saturation. The highest adsorption capacity of CYN by inactivated (83%) and living yeast (63%) was obtained at initial concentrations of 50 and 40 µg L− 1, respectively. This means that each gram of heat-inactivated yeast would remove 455 µg CYN, while the amount of toxin removed by viable yeast would be 264µg toxin g− 1 yeast. Given that the highest level of CYN reported in the aquatic environment is about 100 µg L− 1 (WHO 2020), and the World Health Organization recommended a maximum allowable level of 3 µg L− 1 CYN in drinking water (WHO 2020), the dose of heat-inactivated yeast required for CYN removal to below the proposed guideline value would be 200 mg L− 1.It is noteworthy in our study that CYN binding to inactivated yeast was very stable. Conversely, about 74% of CYN bound to living yeast released back into the washing solution, indicating CYN binding to living yeast was not stable. Previous studies also demonstrated the firmness of mycotoxin and microcystin adsorption to heat-inactivated yeast (Luo et al. 2015; Baptista et al. 2004; Mohamed et al., 2020). Those authors attributed the capability of yeast for toxin adsorption to β-D-glucans present in the yeast cell wall.
In conclusion, this study clearly demonstrates the capability of A. pullulans for inhibiting C. raciborskii growth. Taken the selective antagonistic activity of A. pullulans against cyanobacteria rather than other eukaryotic algae reported in our earlier study (Mohamed et al. 2020), living cells or filtrate of A. pullulans would be employed to selectively control C. raciborskii growth in the aquatic environment. However, biological control measures are still needed to evaluate the feasibility of antagonistic yeast for application in the natural environment. Furthermore, the heat -inactivated cells of A. pullulans had a firmer and higher adsorption of CYN toxin than living cells. Such inactivated yeast would be used to eliminate CYN in drinking water treatment plants as it is non-toxic, pathogenic or infective. Nevertheless, urgent in situ study is needed to commercialize the utilization of heat-inactivated yeast as an adsorbent.