Effectiveness of the toxin-degrading strain screening strategy: Deciency and improvement in screening the gossypol-degrading microorganism Aspergillus niger

The gossypol, as an antinutritional factor or natural plant toxin, has antifertility effects on humans and animals, exists in cottonseed oil and meal, and is a typical food or feed contaminant. It is the classic screening method to using acetate gossypol as the sole carbon sources to prepare selective medium and screening gossypol-degrading microorganisms. By the classic screening strategy, many researchers have discovered that some microorganisms, and Aspergillus niger is the most reported microorganism. A strain of A. niger which can grow in an agar solid medium with gossypol as the sole carbon source is isolated from cattle rumen liquid was obtained by classic screening strategy. The effectiveness of the classic screening strategy was veried, by duplicating and verifying the degradation of gossypol by the A. niger. the A. can free gossypol biosorption (TG)


Background
Toxic substances in soil [1], water [2], food and feed [3] can be degraded by microorganisms. Compared with physical and chemical methods, the biodegradation technology has the advantages of safety, e ciency, moderate, environmental friendliness, absence of secondary pollution and low cost and is the most promising treatment method that has been paid attention by researchers. The classic screening method is that [4]: (1) Samples are usually collected from a variety of speci c areas where degrading microorganisms may exist. (2) A certain toxic substance is used as the sole carbon (or nitrogen) and energy sources, and microorganisms are inoculated in an agar solid medium. (3)The degradation effect is judged using the diameter of the hydrolysis circle or the growth state of the microorganism, and the strains that can utilise or degrade the toxin are screened and isolated. Through further study, targeted degrading enzymes are obtained, and this strategy has achieved many successes [5][6][7].
The gossypol, as an antinutritional factor (ANF) or natural plant toxin, has antifertility effects on humans and animals [8]. The gossypol exists in cottonseed oil and meal (CM), and is a typical food or feed contaminant [9]. Through the classic screening method, many researchers have discovered that some microorganisms, such as Aspergillus niger (AN) [10], Candida tropicalis [11], Aspergillus avus [12] and Bacillus subtilis [13], can degrade gossypol, and AN is the most reported microorganism. On the basis of previous screening strategies, AN that can grow in an agar solid medium with gossypol as the sole carbon source is isolated from cattle rumen liquid. The transcriptome of AN is obtained, and relevant genes or degrading enzymes involved in the degradation of gossypol in AN are attempted to be discovered. However, results have not met our expectations. Therefore, the rigor of the traditional screening strategy for toxin-degrading strains is questioned.
This study has con rmed the de ciencies of previous screening strategies for gossypol-degrading microorganisms and expects to design a scienti c and rigorous screening method for toxin-degrading microorganisms on the basis of existing methods.

Methods
Transcriptome sequencing (RNA-seq) and the trait identi cation of AN AN cultured in potato dextrose agar (PDA) was used as the control group, and AN cultured in PDA with 2% acetate gossypol (AG) was used as the experimental group. The Illumina Hiseq TM 2500 high-throughput sequencing technology was used to sequence the transcriptome of AN under normal and 2% AG conditions. Differentially expressed genes (DEG) were screened, and 2% AG response-related genes were analysed using the gene ontology (GO) and the KEGG (Kyoto Encyclopedia of Genes and Genomes) databases for annotation in AN (measured by Shanghai Biotechnology Co., LTD).
The AN and spores were observed using scanning (SEM) and transmission (TEM) electron microscopy [14].

Growth in different media
Solid media were prepared as shown in Table 1, and an equal amount of the AN spore solution was inoculated and cultured at 37 °C for 24 h. The growth of AN was observed and recorded. And after growing AN for 30 h, the media were stained with Lugol's iodine solution [15] in the dark for 1 min, washed thrice with clean water and observed under a normal light source. Preparation of the crude enzyme and the cell wall pellet suspension AN was inoculated in potato dextrose broth, and the crude enzyme and the cell wall suspension were prepared as follows.
(1) After 24 h, the AN fermentation broth was collected (before spore production), subjected to repeated freeze-thaw and crushed using ultrasonic wave (in ice water) until no intact cell structure was found using a microscope.
(2) The fermentation broth was centrifuged at 4 °C and 8000 r/min for 10 min. The supernatant and the fungus precipitate were collected. After washing the fungus precipitate thrice with aseptic water, the precipitate was resuspended to the original volume, and the cell wall pellet suspension was obtained.
(3) The supernatant was ltered using the millipore lter (0.45 μm) [16], and the crude enzyme was obtained and stored at −20 °C for later use.

Removal experiment of AG by AN components
The experiment was designed and carried out in accordance with Table 2. Experimental groups were restored to normal temperature, and AG was added to make the concentration reach 0.5% and shaken well. Samples 1, 2, 3 and 4 were used as experimental groups, and sample 5 was used as the blank control group. Samples were collected before and after standing at 30 °C in the dark for 24 h and stored at −20 °C for testing. Before testing samples should been thawed, shaken and freeze dried. The free (FG) and the total (TG) gossypol were detected using the international standard ISO6866-1985, and the FG and TG removal rates were calculated according to the formula (1).
Where, Rr: Removal rate, C 1 : Initial content of FG (or TG) and C 2 : Rest of FG (or TG)

GO enrichment analysis of differentially expressed genes
The Unigene GO analysis ( Fig. 1) showed that 25 112 results were annotated and divided into three functional groups, namely, biological process (33.52%), molecular function (28.65%) and cell component (37.83%). The 10 most signi cant differences were DNA binding, transcription DNA-templated, zinc ion binding, G2/M transition of mitotic cell cycle, adhesion of symbiont to host, tryptophan biosynthetic process, asexual spore wall assembly, differentially expressed genes are closely related to cell stress resistance, proliferation, tissue repair, adhesion and cell structure [17][18][19]. Results did not clearly re ect or speculate the existence of the gossypol-degrading gene in AN.

Growth status in different media
AN could grow on all media. At the same culture time, the degrees of growth and maturity followed the order: CzA > sugar-free CzA > sugar-free CzA + 0.5% AG > agar, and spores were produced except in agar ( Fig. 2A). The AN in the agar medium was the weakest (Fig. 2A4) followed by sugar-free CzA added with 0.5% AG (Fig. 2A1). Results showed that screening AN by using agar medium added with AG as the sole carbon source was not rigorous.
Lugol's iodine staining Four media were stained with Lugol's iodine after growing AN for 30 h. Results are shown in Fig. 2B. After staining, corresponding bright spots were observed on the growth position of AN, and the surface of media were slightly dented. The result showed AN could secrete agarase to hydrolyse agar and utilise agar as carbon and energy sources.

Morphological changes in AN
Under the gossypol stress, AN had increased hyphae and decreased number of spores compared with AN grown on PDA alone (Fig. 3A). The spores grown on AG were deformed; had blisters on the surface; thin, uneven and damaged cell wall and small ratio of organelles to cell volume (Fig. 3B). Results showed that gossypol had a negative effect on the AN growth and could cause a bubblelike structure to form on the spores' surface and structural injury in the cell wall. However, AN had a certain tolerance to gossypol injury.
Removal e ciency of fungal components on AG AG was added to each group, and the mixture was placed at room temperature for 1 h (Fig. 4).
Flocculation and sedimentation were observed in all experimental groups. The in uence of the inactivated cell wall pellet (Fig. 4B4) was most notable followed by that of the un-inactivated cell wall pellet (Fig. 4B3).
Different lowercase letters indicate signi cant differences amongst treatment groups (P < 0.05) Compared with the control group, the crude enzyme and the cell wall of AN had signi cant removal effects on the FG in the sample (P < 0.05, Fig. 5), and the inactivated cell wall group had the highest removal rate (42.94%). The FG removal rate of each component after inactivation was higher than that before inactivation. The FG removal rates of cell wall groups were higher than that of the crude enzyme groups, but no signi cant difference was observed amongst experimental groups (P > 0.05).
Different lowercase letters indicate signi cant differences amongst treatment groups (P < 0.05) The components of AN had different removal effects on TG in the sample (Fig. 6). The removal rate of each group was low (all < 15%), and the inactivated crude enzyme group had the highest TG removal rate (13.38%). The FG removal rate of each component was higher after inactivation, but no signi cant difference was observed amongst the same component groups (P > 0.05). The crude enzyme group had signi cant differences amongst cell wall groups (P < 0.05), but no signi cant difference was observed between the control and the experimental groups (P > 0.05).

Discussion
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 [15]. This solution has been used to visualise the agarase activity to screen the agarase production by microorganisms on a culture plate [20]. 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 rst 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 toxindegrading 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 signi cant 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. Signi cant 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 [26]. No signi cant 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 [27]. Hyphae in lamentous 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 [28]. 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 [29]. 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 (CO 2 ) [30] in the air even light [31] may be a carbon or energy for some microorganisms. Thus, factors, such as agar, CO 2 , 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 speci c 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 e ciently 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 di cult 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][35][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, CO 2 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.

Conclusions
In this study, AN is found for the rst time to secrete agarase and utilise agar as carbon source and energy source. AN can reduce the FG content by biosorption but cannot degrade the gossypol and reduce the content of the TG. The classical screening method has limitation in screening gossypol-degrading microorganism.

Availability of data and materials
Not applicable.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions CZ, GQS, HW, DJL, YX, YHZ, MJC, LJC designed and analyzed the experiments. C Z and GQS performed the experiments. CZ and LJC interpreted the data and wrote the manuscript, and were major contributors in writing the manuscript. All authors read and approved the nal manuscript.