In this experiment, AN can grow in four kinds of media, and screening the degrading strains simply by using an agar medium added with toxin as the sole carbon source is not rigorous. The Lugol’s iodine solution can stain polysaccharides in agar into a dark brown colour and cannot stain the degraded oligosaccharides of agar . This solution has been used to visualise the agarase activity to screen the agarase production by microorganisms on a culture plate . Similar to the AN in this study, some strains [15, 21] can utilise agar to grow normally on the medium without any sugar, interfere with the expected experimental results and affect the experimenter to make a true judgment on the results.
Enzymes are metabolites secreted by living organisms and can be divided into primary (constitutive enzymes) and secondary (inducible enzymes) metabolites. This study first discovers that AN can secrete a constitutive enzyme that hydrolyses agar and utilise agar as sole carbon source. Therefore, we suspect that the gossypol-degrading enzyme of AN may not be a primary metabolite and that the gossypol cannot be directly utilised. This enzyme may be an inducible enzyme and can be secreted only under the condition of the gossypol as an inducer. In this study, the gossypol has a negative effect on the normal growth of AN. However, AN has a certain tolerance to the gossypol injury and produces active spores. Thus, AN may produce some special inducing enzymes under the induction of the gossypol. Given that the premise of the basic physiological metabolism of the cell is guaranteed, when exogenous stimuli interfere with the normal metabolism of cells or cause cell damage, certain proteins (such as cytokines and inducible enzymes) are secreted abundantly by the cells, which are under the exogenous stimulus stress against exogenous substances and protect cells [22, 23]. Therefore, when screening for a toxin-degrading strain by using a medium with the toxin as the sole carbon (or nitrogen) and energy sources, the function of inducing enzymes may be overlooked by usual screening methods of the predecessors.
For the removal effect of FG (or TG), no significant difference is observed before and after inactivation in crude enzyme groups (or cell wall groups). The TG removal rate of each group is lower than the FG removal rate (Fig. 5 and Fig. 6). For AN, the FG removal function and metabolic enzymes may not be closely related. Heating promotes proteolysis and increases the content of free amino acids [24, 25]. The crude enzyme solution is composed of proteins, and the cell wall suspension also contains some little cell wall proteins. After heat inactivation, the content of free amino acids in the solution increases. The Maillard reaction occurs and is strengthened between the carbonyl group from the gossypol and free amino acids under ISO detection conditions. As a result, the TG content decreases, and the TG removal rates of the crude enzyme and the cell wall groups increase when inactivated. Significant differences between the crude enzyme and the cell wall groups are observed due to different protein contents. The gossypol is unstable at room temperature, and a small amount of gossypol is degraded . No significant difference in the TG removal rate is observed between the control and the crude enzyme or the cell wall group (P > 0.05). Thus, we believe that the decrease in TG content is not due to AN.
Hyphae secrete enzymes that convert the organic material from media into small molecules that can be taken up by the fungus to serve as nutrients . Hyphae in filamentous fungi also serve as store house for nitrogen and phosphorus in the form of DNA, giving hyphal tips the capability of persistent extension and foraging in new areas . The inadequate nutrition in sugar-free medium prompts AN to increase absorbable nutrients indirectly by increasing the number of hyphae. The toxic effect of the gossypol prompts AN to extend away from the gossypol damage by increasing the number and the length of hyphae. The agar medium lacks minerals and is not enough to support the growth of AN. AN relies on stored nutrients in spore growth and grows weakly. The main components of the AN cell wall are chitin and glucan and used as biosorbent for heavy metals . The removal effect of AN on FG may be due to the biosorption of fungi. The gossypol can destroy the structure of AN and increase the gene expression of fungus cell stress resistance, proliferation, tissue repair, adhesion and cell structure. The reduction in gossypol is more likely to be consumed by damaging AN or protein.
When screening toxin-degrading microorganisms, we usually regard the toxin as the sole carbon source, in the preparation of agar solid medium tend to ignore the agar could be a potential carbon source, moreover, in culturing, is likely to ignore the carbon dioxide (CO2)  in the air even light  may be a carbon or energy for some microorganisms. Thus, factors, such as agar, CO2, illumination and inducible enzymes should be fully considered.
The degradation effect of microorganisms on toxin is an important basis to evaluate whether strains have further research value. Toxins can be absorbed by microbial adsorption, and free toxins are remarkably reduced. but no degradation, will free again under specific conditions, when evaluating the toxin degradation effect of a strain, the reduction binding toxins should be considered fully, basing on the change in total toxin to determine whether the target strain adsorbs or degrades toxins.
Besides, substances or toxins can be directly, exactly and efficiently analysed quantitatively and qualitatively by using a mass analyzer and other equipment [32, 33]. The toxin can be more intuitive judged whether or not to be degraded or form a new substance. Yet the toxin degradation products by microorganisms usually have many characteristics, such as low content and mixed with a variety of complex substances, the composition is complicated and difficult to extract and purify. In addition, the mass analyser has high requirements on operating ambient detection conditions and is not suitable for use at the initial phases of screening degrading strains.
During the processes, such as agricultural production, the exploitation and the utilisation of resources, sewage treatment and food storage and processing. Various degrees of toxin pollution, including organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), dyes, mycotoxins and others, are available, and most of them are refractory organic pollutants, bioaccumulative, persistent and toxic with carcinogenic and mutagenic activities [34–36]. The successful screening and the domestication of microorganisms with degradation effect on a certain pollutant are important for environmental protection and human health. When screening toxin-degrading strains by using a toxin as the sole external carbon source and the solid medium as gel, the selection strategy should be optimised, and control groups with agar, CO2 and light should be established at the same time. The screening scope can be extended to toxin-resistant organisms, and the content can be reduced by the inducible enzyme or consumed by the injury of resistant organism. The screening of pollutant-degrading strains is a tedious task and contingent in nature. Therefore, screening strategies should be fully considered when conducting strain screening for improved accuracy.