4.1 Identification of the dominant microbial groups associated with the CSD
Microbes have been recognized as important and critical contributors to OMD in rivers [9, 16]. Invertebrates can also decompose organic matter, but their contributions are usually influenced by microbial conditions (e.g., providing nutrients and palatability). The relative importance of invertebrates and microbes in OMD may vary depending on the location and conditions of rivers . We did not find macroinvertebrates on CS during sampling in this study, which was consistent with the results of the preliminary experiment. This implied a fairly low possibility of CSD by macroinvertebrates. Simultaneously, the influences of the hydraulic shear force and abrasion on CSD were systematically reduced or even eliminated by placing CS at positions with consistent and slow flow velocity and not in contact with the sediment. Therefore, the CSD that occurred in rivers was most probably due to the enrichment of bacteria and fungi groups related to cellulose decomposition by the CS.
The CS is mainly composed of cellulose (≈95%), which is a key carbohydrate on earth and a major basal resource in most of Earth’s food webs . The hydrolysis of cellulose into smaller molecules that can be assimilated is achieved mainly through cellulases synthesized by cellulose decomposing bacteria and fungi. Cellulases mostly include endoglucanase (EndoG), exoglucanase (ExoG) and β-glucosidase (β-Glu), integrating to achieve the decomposition of cellulose . The six shared dominant bacterial phyla and two shared dominant fungal phyla observed in the present study have been reported as the dominant phyla on cellulose-rich media [56-59]. In order to reveal their role in the CSD, RDA was conducted, respectively. In Fig. S3 and S4, all of the canonical axes significantly explained the variations of CSD, with an explanation rate of 31.38% for bacteria (P = 0.008) and 28.15% for fungi (P = 0.010), respectively. This indicated the shared dominant bacterial and fungal phyla were significantly associated with CSD, respectively. Bacterial phyla Proteobacteria was significantly related to CSD with an explanation rate of 12.2% (P = 0.013). Rozellomycota and Ascomycota were the significant fungal phyla related to CSD with the explanation rate of 16.4% (P = 0.010) and 12.1% (P = 0.012), respectively. These three phyla were positively correlated with TSL, indicating their contributions to TSL. Phyla Proteobacteria [60-62], Rozellomycota [59, 62] and Ascomycota [63-65] have been reported previously to produce cellulase or be detected in cellulose-rich media. Moreover, Ascomycota and Rozellomycota were the aerobic and anaerobic fungal phyla reported in previous studies [59, 66]. Ascomycota and Rozellomycota were positively and negatively correlated with RES in this study, indicating they increased and decreased RES, respectively. The aforementioned discussions suggested that CS in rivers could selectively enrich bacteria and fungi groups related to cellulose decomposition, thus resulting in the CSD in rivers.
4.2 Cooperation pattern of bacteria and fungi and contributions of keystone taxa and key modules to CSD
Bacteria and fungi on CS interacted to form a microbial network to achieve the CSD. The network constructed by bacterial and fungal genus had 662 nodes and 4411 edges in this work, indicating that the bacterial and fungal genus on CS were well integrated with each other and formed a co-occurrence network. This was also found in previous studies on microbial co-occurrence patterns in organic-rich soils . One possible explanation was that a great supply of nutrients from CS and river water might release microbes from resource limitation, stimulate microbial growth, and provide more opportunities for interactions among different taxa [67, 68]. The number of F-F edges was the lowest in our network, indicating that fungi linked with more free-living or less connected in the network compared to bacteria . But, their roles on OMD cannot be ignored in aquatic environments, because recalcitrant organic matters like plant litters were mainly decomposed by fungi . The highest number of B-F edges indicated the close interactions between bacteria and fungi, which are consistent with their ecological associations and common in microbial community. . The number of positive correlations was much higher than that of negative correlations between bacteria and fungi in the network (Fig. 6a), indicating the cooperation of bacterial and fungal genus in CSD. The products such as low molecular weight organic matter released in the decomposition process of recalcitrant organic matters by fungi can be used by bacteria [12, 22]. Meanwhile, nutrients activated by bacteria can facilitate their absorption and utilization by fungi, and bacterial metabolites can also stimulate the growth of fungal hyphae . The synergistic relationship between fungi and bacteria can also be due to the close spatial relationship between them, with bacteria as epiphytes on fungi , which deepened the interaction between bacteria and fungi.
The high modularity of microbial network was conducive to the stability of microbial community structure, function and micro-ecosystem, so as to resist the drastic changes of environments . In this study, the network presented obvious modularity and was divided into 61 modules. There were differences in the groups and quantities of bacteria and fungi involved in different modules. This was probably because of the functional specificity of modules, depending on the combinations of bacteria and fungi . The module has also been considered as the niche, and modules identified in the network reflect the environmental preference and habitat heterogeneity of microbes . Although changes in environmental conditions may lead to variations in certain microbes in the module, they can be replaced by microbes with similar ecological characteristics or functional roles on ecosystem stability . Thus, the module is usually relatively stable and preserves its necessary roles in sustaining the ecosystem functions . In our network, most of the interactions within modules were predominantly positive. This suggested that microbes within the same module may form cooperative interactions or share similar niches . Meanwhile, there were some cross-module edges, which were mainly positive, indicating that the modules in this network were not isolated from each other but cooperated with each other. It has been reported that nutrient availability was the important shaping factor in network modules . During the cellulose decomposition process, different and bioavailable nutrients are continuously released, providing various nutrient niches to form different modules. This thus resulted in positive correlations among the modules.
Two key modules were obtained in this study, i.e., modules 2 and 4, which were significantly positively correlated with RES and TSL, respectively. This indicated that members of the two key modules might have their own special functions to contribute to CSD. It was worth mentioning that the bacteria and fungi in the two key modules cooperated to promote RES, because the edges in the key modules were mainly B-F (44.83%) and were predominantly positive. There were 41 bacterial genera and 22 fungal genera involved in the key module 2, such as bacterial genera Actinotalea, Armatimonas/Armatimonadetes_gp1 and Methylobacter, as well as fungal genera Sampaiozyma and Cystofilobasidium. They were typically reported as aerobic or facultative anaerobic [76-79]. Most of them belonged to the shared dominant phyla Proteobacteria and Ascomycota, which were positively correlated with RES (Fig. S9 and S11). Module 4 included 31 bacterial genera and 13 fungal genera, such as bacterial genera Flavobacterium and Phaeodactylibacter, and fungal genera Geotrichum and Exophiala. They have been reported to be related to cellulose decomposition [79, 80]. Most of these bacteria and fungi belonged to the shared dominant phyla Proteobacteria and Ascomycota, which were positively correlated with TSL (Fig. S9 and S11), indicating they could synergistically increase TSL. The key modules 2 and 4 positively direct interacted with other modules in the network, indicating that the key modules may connect with other modules through exchanging the nutrients during the cellulose decomposition process, and thus formed a bacterial-fungal cooperative network to achieve the CSD.
Keystone taxa in the network represented the critical regulators driving the microbial interactions . Keystone taxa regulate the community structure and function mainly through acting on the intermediate or effective groups by secreting metabolites, antibiotics and toxins irrespective of their abundance [23, 81]. He et al. emphasized that the keystone taxa might develop strong relationships with other members in the module through antibiotics or secondary metabolites . In this study, 14 bacterial genera and 7 fungal genera were detected as keystone taxa, but their relative abundances were not high. This was consistent with recent microbial network studies in which low abundance taxa might act as the keystone taxa in other different environments [73, 83-86]. These keystone taxa with less abundant may play important roles in determining genetic diversity, functional diversity, and ecosystem stability . Moreover, there were close cooperative relationships among these keystone taxa because all 44 edges were positive. We found that the keystone taxa and the modules containing keystone taxa had no significant correlation with CSD. However, some keystone taxa had direct interactions with the members in key modules 2 and 4. This suggested that the role of the keystone taxa in CSD may be more reflected in their connections with some species contributing to CSD in the key modules, rather than directly decomposing CS. These keystone taxa were very important to understand the CSD caused by the bacterial-fungal network. Removing these keystone taxa may catastrophically disrupt the bacterial-fungal network into many disconnected subnetworks and dramatically decrease the ecological functions at the community level . Taking above together, the bacterial-fungal interactions were dominated with cooperation, reflecting in the keystone taxa, within the key modules, as well as between the key modules and others. The role of keystone taxa was to connect with the microbial groups associated with CSD in key modules, and then the key modules directly promoted the CSD.
4.3 Regulation of the dominant environmental factors to keystone taxa and key modules
Exploring the relationships between the dominant environmental factors and key modules, keystone taxa in the bacteria-fungal network on CS will help to better understand how these microbial interactions are affected by their habitat conditions. Mantel test results showed that key module 2 was significantly positively correlated with water temperature, and key module 4 was significantly positively correlated with TN. Microbes with a similar preference for water temperature and TN tended to gather together to form key modules 2 and 4, respectively, and in turn, these formed modules were susceptible to changes in water temperature and TN. Temperature affects the enzymatic reactions in microbes and is an important control factor for the growth and metabolism of microbes. Nitrogen is a restrictive element of microbes in environments. It plays an important role in the life and metabolism of microbes by affecting protein synthesis, cell division, etc., processes . It has been widely reported that temperature and TN were the key regulatory factors for the assembly of microbial communities in other environments, and they strongly adjusted microbial interactions [89-91]. Previous studies about soil environments have documented that the modules in microbial networks were significantly positively correlated with environmental temperature and nitrogen concentration [22, 70, 92, 94], which provided support for our results. Two keystone taxa (i.e., Emticicia and Flavihumibacter) in module 1 were significantly positively correlated with TN in this study. This indicated environmental nitrogen concentration might regulate the stability of microbial network through acting on the keystone taxa preferentially, finally achieving the CSD. Similar results have also been confirmed in other studies in soil environments [86, 91, 95]. A large number of previous studies have reported that water temperature and TN were important factors affecting the OMD in river ecosystems [8, 10, 32]. Our results provided the important mechanisms for those previous studies, i.e., water temperature and TN promoted the OMD in river ecosystems through regulating the keystone taxa and key modules in the bacterial-fungal network.