Effect of inoculant and fungal infection on the fermentation profiles and mycotoxins of corn silages during aerobic exposure
Once the silage is opened and fed, air freely accesses the silo face and aerobic microorganisms that survived the ensiling process, e.g. bacilli, yeast and acid-tolerant bacteria, can rapidly proliferate, metabolizing residual sugars and organic acids to CO2, H2O, and heat [14, 15]. Consequently, silage temperature increases and the silage mass becomes aerobically unstable. The consumption of acids by aerobic microorganisms is accompanied with the increase of silages pH, thus, the variation of silage pH is used as the criteria of aerobic deterioration [16]. In the study, the pH of NFI silages showed a slight increase within 2 d followed by a marked increase, while that in FI silages rapidly increased during the initial 2 d of aerobic exposure, this might be attributed to the rapid revival of aerobic microorganisms including infected toxigenic fungi [16]. This hypothesis was confirmed by the changes of LA: the LA in FI sharply decreased once aerobic exposure, while the rapid drop of LA was following a transient stable (2 d) in NFI silages. Kung, et al. [17] also reported that the increase of silage pH was accompanied by a decline of LA in corn silages during aerobic exposure. The faster and larger changes of pH and LA in FI than NFI silages were related to its lower AcA concentration. The AcA has been proven to be responsible for enhancing aerobic stability because it acts as an inhibitor of spoilage organisms. Danner, et al. [18] stated that exponentially increased silage stability with AcA concentration were attributed that the lipophilic and undissociated form (around pH 4) of AcA, which could penetrate the bacterial plasma membrane and disorder metabolism in cell. Queiroz, et al. [19] also found that fungal infection worsens the fermentation of corn silage, resulted the lower AcA production during ensiling, which might be partly contributed the faster and larger changes of pH and LA in FI than NFI silages in the present study.
Although other modes of action may exist, the production of AcA has been the most accepted explanation of how organisms from the Lb. buchneri group of bacteria increases the aerobic stability of silages [7]. In the study, the higher AcA concentrations in LB and LBLP than other treatments of NFI silages lasted to d 2 and 4, respectively, while no significant difference in AcA concentrations was observed among FI silages. This indicated that the fungal infection might disturb the microbial community and fermentation of silages, diminishing the effect of LB on the AcA production. The decrease of WSC decreased with prolonged aerobic exposure was attributed to the extensive proliferation of aerobic microorganisms, which can oxidize WSC to CO2 and H2O [20]. FI silages always had lower WSC concentrations than NFI silages. It can be conjectured that infected fungi consumed more WSC during the initial transient aerobic stage of ensiling, resulting in the lower residual WSC being in FI silages than NFI silages. For NFI silages, the concentration of WSC in LB and LBLP was consistently higher than that of C and LP over the 6 d of aerobic exposure. This was attributed to the more AcA produced by L. buchneri, which inhibited the proliferation of undesirable microorganisms, preserving more residual WSC [21]. Kung, et al. [22] reported that higher dissociation constants (pKa) of AcA (4.75) than LA (3.86) contributed to the higher antimicrobial activity of AcA in surroundings where the pH values are low (around pH 4), since a greater proportion of the acetate is undissociated status, which could pass through the bacterial or fungal cell membrane and release protons to acidify the cytoplasm, thereby inhibiting or killing microorganisms [23, 24]. In the study, there was a transient increase in the ethanol concentration of FI silages, which was possibly due to the metabolism of yeast within the initial 2 d of aerobic exposure [25]. FI silages had higher number of yeasts and molds than NFI silages. Irrespective of fungal infection, the lower numbers of yeast and molds in LB and LBLP than C and LP on d 2 and 4 were attributed to the fungistatic property of AcA produced by the inoculants of L. buchneri [2]. With the progress of aerobic exposure, the gradual decline of ethanol concentration might be due to its volatilization or metabolized by Acetobacter [26].
Effect of inoculant and fungal infection on the bacterial and fungal communities of corn silages
During aerobic exposure, application of LAB reduced the Shannon and Chao 1 indexes of bacterial and fungal communities in NFI silages, but not FI silages. This might be due to the proliferation of aerobic bacteria and yeasts, which occupied the predominant roles of the microbial community [20].
In the study, Acetobacter became the dominant bacteria after 6 d of aerobic exposure, because it can oxidize ethanol to AcA initially, followed by oxidation of LA and AcA to CO2 and H2O [27]. Acetobacter is non-fermenting aerobic bacteria and can be found in various environments, it is able to initiate aerobic deterioration of corn silage with or without the presence of yeasts [28]. Guan, et al. [29] also observed that the significant increase in the RA of Acetobacter in Napier grass after 2 d of aerobic exposure. Lactobacillus is the main bacteria involved in LA fermentation during ensiling and usually dominates the well-fermented silages [30], however, it is rapidly replaced by aerobic bacteria (e.g. Acetobacter spp.) once silage is exposure to air [31]. In the present study, the RA of Lactobacillus reduced below 1% in silage except C and LB of NFI silages after 6 d of aerobic exposure. The genus Klebsiella and Enterobacter, belonging to enterobacteria, are capable to produce ammonia, which can cause animal health issues [32]. In the study, the decline of RA of Klebsiella and Enterobacter might be attributed to the anaerobic property and intolerance to acid conditions, as reported by McGarvey, et al. [33], who found Enterobacteriaceae could thrive in anaerobic and weak acidic conditions (pH > 5.4). In the study, the silage pH was not increased up 5.4 until d 6 of aerobic exposure, which resulted in the substitution of Klebsiella and Enterobacter by Acetobacter. The higher RA of Acinetobacter in NFI than FI silages might be related to the higher AcA concentration, which could be used by Acinetobacter as substrate [34].
Issatchenkia, an acid-tolerant yeast, is the dominant fungal genus in NFI silages regardless of aerobic exposure or inoculation. However, there are marked variations in fungal composition for FI silages during 6 d of aerobic exposure. This discrepancy between NFI and FI silages was attributed to the fungal infection, which resulted in more yeast and molds present and revived in FI silages during aerobic exposure. For FI silages, the marked decline in the RA of Issatchenkia was accompanied by the increase of RA of Kazachstania and Zygosaccharomyces during 6 d of aerobic exposure. Wang, et al. [35] indicated that Kazachstania had a strong tolerance to LA and was crucially involved in initiating the aerobic deterioration of corn silage with a relatively low pH and AcA content. Hao, et al. [36] reported that Zygosaccharomyces bailii was the sole yeast species isolated from spoilage total mixed ration (TMR) silages and confirmed that the Z. bailii could initiate aerobic deterioration of TMR silages. In the study, the RA of Candida and Pichia decreased below 1% after 6 d of aerobic exposure, this is contrast to the report by Duniere, et al. [37], who found that Candida and Pichia were the main spoilage genera after aerobic exposure. Pahlow, et al. [38] indicated that Issatchenkia, Candida, and Pichia are lactate-assimilating yeasts that the initiators of aerobic degradation of silage. We speculated that Kazachstania and Zygosaccharomyces underwent more vigorous growth and outcompeted other yeast and molds.
The correlation between microbial community and fermentation profiles was analyzed by RDA. In the present study, bacterial genera such as Lactobacillus were found to be positively correlated with LA and AcA, while Acetobacter was negatively correlated with LA and AcA regardless of fungal infection. When silage is exposed to air, LA and AA were metabolized by aerobic bacteria, increasing silage pH, which further inhibited the proliferation of Lactobacillus and boosted the dominance of Acetobacter [39]. Fungal genera Kazachstania and Zygosaccharomyces were negatively correlated with LA and were positively correlated with pH. This indicated that Kazachstania and Zygosaccharomyces were considered as dominant fungi during aerobic exposure of silages, which could assimilate lactate and increase pH, initiating the spoilage of silages.
Effect of inoculant and fungal infection on mycotoxins of corn silages during aerobic exposure
In the study, the concentrations of most mycotoxins increased over 6 d of aerobic exposure, indicating that toxigenic fungi revived during the aerobic exposure [40]. The larger increases of AFs, ZEN, and DON concentrations in FI than NFI silages confirmed that artificially infected A. flavus and F. graminearum might revive and produce mycotoxins during the aerobic exposure stage. Ferrero, et al. [41] indicated that the aerobic environment allowed proliferation of A. flavus and enhanced the production of AFB1. Vandicke, et al. [42] also reported that some Fusarium species were even able to survive at low oxygen levels (< 0.5%), such as silage conditions, and the inactive fungal spores in the silage may be reactivated and produce Fusarium toxins due to exposure to oxygen during feed-out.
The more effective of inoculants on reducing AFB1 contamination in NFI than FI silages might be attributed to the revival of toxigenic fungi and higher mycotoxins in FI silage, which attenuated the effects of inoculants on reducing AFB1 contamination of FI silages. Ma, et al. [9] reported that L. plantarum, L. buchneri, and Pediococcus acidilactici could bind to AFB1 in an in vitro medium. Dogi, et al. [43] suggested that inoculation with Lactobacillus rhamnosus strongly inhibited the fungal growth (F. graminearum, Aspergillus parasiticus, etc.) and mycotoxin production (AFs, ZEN, DON, etc.). However, it is unexplainable that the AFB2 in LB of FI silage was decreased during aerobic exposure in the study.
Effect of inoculants and fungal infection on aerobic stability of corn silages
The fungal infection shortened aerobic stability of silages regardless of inoculation. This was attributed to the artificial fungal infection, which resulted in more yeast and molds survived in FI than NFI silages. Keshri, et al. [44] reported that the lower aerobic stability of corn silages was due to the high number of lactate-assimilating yeasts. In the study, the aerobic stability of NFI silages was improved by the application of LB and LBLP as compared with C of NFI silages. Kung, et al. [17] also found that inoculant containing L. buchneri could extend the aerobic stability of silages challenged with air stress, which was attributed to the antifungal property of AcA produced by L. buchneri. Romero, et al. [45] reported that applying a combination inoculant of L. buchneri and Pediococcus pentosaceus increased the AcA concentration and decreased yeasts and molds in corn silages, which in turn indirectly extended the aerobic stability.