Li Jia

Qing Liu

Siyi Chen

Kexue Liu

Yiqing Chen

Mikael Motelica-Heino

Hesen Zhong

Menghao Zhang

Cevin Tibihenda

Patrick Lavelle

Chi Zhang

Jun Dai

Brown George

Weixin Zhang
email: weixinzhang@139.com

Manuel Blouin
email: manuel.blouin@agrosupdijon.fr

Keywords: Earthworm, Sphingobacterium sp., Cd-contaminated soil, carbon and nitrogen forms, enzyme activities

published or not. The documents may come

Individual and combined effects of earthworms and Sphingobacterium sp. on soil organic C, N forms and enzyme activities in non-contaminated and Cd-contaminated soil

Answer: Thanks for your comment. We added more detail about earthworm biomass after checking and confirming the data as "Each pot was inoculated with 3 g of earthworms, approximately 8 individual E. fetida (~ 0.37 g•ind-1) or 5 individual A. gracilis (~ 0.60 g•ind-1).". In previous study, the average weight of E. fetida is around 0.4 g•ind-1 (Yue et al., 2019). Therefore, we think it is in normal range of biomass of E. fetida. Reference: S.Z. Yue, H.Q. Zhang, H.Y. Zhen, Z.Q. Lin, Y.H. Qiao, Selenium accumulation, speciation and bioaccessibility in selenium-enriched earthworm (Eisenia fetida), Microchemical Journal 145 (2019) 1-8, https://doi.org/10.1016/j.microc.2018.10.015. 4. Line 173, What is DTPA Cd, and how to measure it? Answer: Thanks for your comment. We added more details about DTPA and measure method in lines 203-213 as: 2.4 Total and DTPA extractable Cadmium content Total cadmium (Cd) in the soils was extracted by microwave digestion with aqua regia (HNO3:HCl (v/v) = 1:3). The diethylenetriaminepentaacetic acid (DTPA) extractable form was regarded as bioavailable metals and toxic. DTPA-extractable Cd was extracted with DTPA extractant at a Soil:DTPA extractant ratio of 5:25 (g/mL), which included a solution of 5 Mm DTPA and 10 mM CaCl2. The extracted suspensions were analyzed for total cadmium (Cd) and DTPA-extractable contents using inductively coupled plasma-mass spectrometry (ICP-MS, 7700ce, Agilent Technologies, Santa Clara, CA, USA) [43]. GBW07455 (GSS-26) was employed as certified reference material for soil analyses, and the measured concentrations were within 98% of the reported certified concentrations for Cd. Quality control measures included three repetitions for each soil and three blanks for each batch to ensure the quality of the analysis. 5. Lines 322-326, Not sure here, a lower standing MBC did not necessarily indicate a low growth of bacterial population, alternatively, this may also result from a strong feeding of earthworm on the bacterial biomass? Answer: Thanks for your comment. We agree with you. The feeding of earthworm also strong impacts the microbial biomass and structure. We revised and improve this part as: Line 402-411: Earthworm activities under the combined process dramatically decreased microbial biomass carbon compared to Sphingobacterium sp. activities alone. It is suggested that earthworms contribute to maintaining the ecosystem balance and stabilizing the microbial community structure through feeding [START_REF] Zhang | Influence of the earthworm Pheretima guillelmi on soil 676 microbial biomass and activity[END_REF] and regulating the allocation of active organic carbon and dissolved organic carbon pools [START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Yu | Effects of straw application and earthworm inoculation on 679 soil labile organic carbon[END_REF]. Therefore, the rapid proliferation of Sphingobacterium sp. was prevented in CSE and CAS treatments. It was further confirmed by the significantly lower microbial quotient carbon in treatment CSE and CSA compared to treatment CS (Fig. 2E). It can be deduced that earthworms may reduce the conversion efficiency of soil microorganisms in utilizing carbon sources into microbial biomass carbon [52] or they can intensely feed and graze on microbes [10,[START_REF] Vafa | Sewage sludge application strongly modifies earthworm 681 impact on microbial and biochemical attributes in a semi-arid calcareous soil from Iran[END_REF][START_REF] Edwards | Interactions between earthworms, microorganisms, and 684 other invertebrates[END_REF][START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF]. 6. Lines 355-356 and lines 414-415, so, how to understand such negative effects of earthworms? similar negative effect was also reported in literature, such as Lv et al., 2020 https://doi.org/10.1016/j.scitotenv.2020.137227; Answer: Thanks for your comment. Likewise, in [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF], earthworm activities suppressed β-glucosidase activity during stimulating carbon mineralization processes. They speculated that when soil microorganisms could easily access the readily mineralized C released by earthworms, they would use a smaller portion of the carbon from enzyme-catalyzed decomposition of soil organic carbon. Additionally, the enzyme activity only represents the concomitant consequence of earthworm activities but does not serve as the major driver of organic matter decomposition. It is also controlled by multiple factors such as food type, pH, temperature, gut transit time, metal stress, substrates, et al., [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF][START_REF] Karasov | Ecological physiology of diet and digestive 661 systems[END_REF][START_REF] Utobo | Soil enzymes as bioindicators of soil ecosystem status[END_REF]. We revised the discussion of negative effects as: Line 358-365 as: As for enzymes, the enzymes activity was influenced by earthworm species and metal stress. Likewise, in the previous study, earthworm activities suppressed β-glucosidase activity during the stimulation of C mineralization processes [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF]. It was speculated that when soil microorganisms could easily access the readily mineralized C released by earthworms, they would use a smaller portion of the carbon from enzyme-catalyzed decomposition of soil organic carbon. Enzyme activity only represents the concomitant consequence of earthworm activities but does not serve as the major driver of organic matter decomposition. It is also controlled by multiple factors such as food type, pH, temperature, gut transit time, metal stress, substrates, et al., [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF][START_REF] Karasov | Ecological physiology of diet and digestive 661 systems[END_REF][START_REF] Utobo | Soil enzymes as bioindicators of soil ecosystem status[END_REF]. 7. lines 414-415 may suggest that soil microbiota did not play a key role in OC decomposition processes, but changed accordingly, worm dominated these processes. Answer: Thanks for your comment. You are right. The soil enzymes activities shown inhibited which may suggest that the earthworm dominated the organic carbon decomposition processes and Sphingobacterium sp. play an assistance role. Another underlying factor is that Sphingobacterium sp. may assist earthworms in mineralizing soil organic carbon and nitrogen by stimulating the microbial activities in the earthworm gut. The subsequent studies need to focus on the earthworm gut microbial to reveal the internal mechanisms. We have added some discussion in the manuscript as: Line 393-417: In these processes, earthworm activities dominated the soil carbon and nitrogen mineralization processes, while Sphingobacterium sp. played a supporting role in combined processes. When the effects of earthworm and Sphingobacterium sp. on carbon and nitrogen were consistent, they enhanced each other; otherwise they undermined each other. Hence, in non-contaminated soil, carbon mineralization was observed. While in Cd-contaminated soil, carbon storage was more prevalent due to the stronger effect of earthworms on stabilization than Sphingobacterium sp. on mineralization. This is further confirmed by PCA which demonstrated that earthworms play a critical role in soil organic carbon decomposition processes, following the same trend as inoculation with earthworm alone and in combination with Sphingobacterium sp. (Fig. 4A andC; Fig. 5A andC). Additionally, in this system, a strong interaction existed between earthworms and Sphingobacterium sp.. Earthworm activities under the combined process dramatically decreased microbial biomass carbon compared to Sphingobacterium sp. activities alone. It is suggested that earthworms contribute to maintaining the ecosystem balance and stabilizing the microbial community structure through feeding [START_REF] Zhang | Influence of the earthworm Pheretima guillelmi on soil 676 microbial biomass and activity[END_REF] and regulating the allocation of active organic carbon and dissolved organic carbon pools [START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Yu | Effects of straw application and earthworm inoculation on 679 soil labile organic carbon[END_REF]. Therefore, the rapid proliferation of Sphingobacterium sp. was prevented in CSE and CAS treatments. It was further confirmed by the significantly lower microbial quotient carbon in treatment CSE and CSA compared to treatment CS (Fig. 2E). It can be deduced that earthworms may reduce the conversion efficiency of soil microorganisms in utilizing carbon sources into microbial biomass carbon [52] or they can intensely feed and graze on microbes [10,[START_REF] Vafa | Sewage sludge application strongly modifies earthworm 681 impact on microbial and biochemical attributes in a semi-arid calcareous soil from Iran[END_REF][START_REF] Edwards | Interactions between earthworms, microorganisms, and 684 other invertebrates[END_REF][START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF]. Additionally, the different impact of earthworm species may be caused by E. fetida being more conducive to the survival and growth of G-bacterial communities [START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF], thus promoting Sphingobacterium sp. to assist earthworms in carbon mineralization. Based on the above, another underlying factor was postulated that Sphingobacterium sp. may assist earthworms in mineralizing soil organic carbon and nitrogen by stimulating the microbial activities in the earthworm gut. The subsequent studies need to focus on the earthworm gut microbial to reveal the internal mechanisms. 8. lines 392-393, how could the assistance role of Sphingobacterium sp. on worm could be observed in Fig. 4a? Answer: Thanks for your comment. Sorry for our mistakes. This conclusion cann't be observed only in Fig. 4A, it should be analysis through combining Fig. 4A and Fig. 4C. The low PC1 values in Fig. 4A means high carbon and nitrogen mineralization such as DOC and AN. Inoculation earthworm treatments trend to have low PC1 values especially co-inoculation with Sphingobacterium sp. (Fig. 4C). It means that coinoculation with Sphingobacterium sp. can enhance the earthworm effects on carbon and nitrogen transformation. We added the details of figure label as: Line 399-401: This is further confirmed by PCA which demonstrated that earthworms play a critical role in soil organic carbon decomposition processes, following the same trend as inoculation with earthworm alone and in combination with Sphingobacterium sp. (Fig. 4A andC; Fig. 5A andC).

Reviewer #2: Jia et al. investigated the effect of single and combined application of earthworms and Sphingobacterium sp. on the C and N cycles in a non-contaminated soil and a cadmium-contaminated soil. The C and N fractions in soil, ratio of C/N fractions, soil enzyme activities, relationships among the above parameters were determined. The results of this study can provide a better understanding of how different species of earthworms and their co-application with a key bacterium from the gut of earthworm regulate the carbon and nitrogen mineralization/fixation in the non-contaminated and Cd-contaminated acid soils. Yet, the current version of the manuscript still needs Answer: Thanks for your comment and suggestion. We added the effects of inoculation with Sphingobacterium sp. alone. Like below: "Sphingobacterium sp. could promote C mineralization in both soils.". 3. Introduction. 1) What is the hypothesis for the potential effect of Sphingobacterium sp. on carbon and nitrogen cycling process in Cd-contaminated soil? The authors need to add more background information to support their hypothesis in the "Introduction" part. Answer: Thanks for your comment. We rearrange the introduction part and supply the background information and the potential effect of invocation Sphingobacterium sp. alone as: Line 108-112: However, under environment stress, such as metal contamination, the activities and complex interactions of them may be suppressed. Specifically, metal contamination can induce biomass loss, reduce survival rate, cause irreversible physiological damage, decrease the stability of gut bacterial network, and dampen its ecological functions [27][28][29]. Similarly, the inhibition effects also impact soil microorganisms, leading to reduced soil enzyme functions critical for organic matter cycling [30]. We also added the hypothesis in as: Line135-140: Given that Sphingobacterium sp. is the dominant bacterial taxon in the earthworm gut, we hypothesized that Sphingobacterium sp. and earthworm could individually promote soil organic matter decomposition and, when combined, exert reinforcing effects. These effects are negatively impacted by metal stress and influenced by the survival strategies of different earthworm species; Additionally, Sphingobacterium sp. and earthworms may play different roles in this combined process, with Sphingobacterium sp. stimulating microbial activities in the earthworm gut.

2) The specific objectives were not consistent with the "abstract" and "discussion". Answer: Thanks for your comment. We revised the specific objectives as: Line 143-148: The specific research objectives were (1) to analyze and compare the effects of Sphingobacterium sp. and the two earthworm species, when inoculated alone and in combination, on the carbon and nitrogen forms and related enzyme activities in non-and Cd-contaminated soils. (2) to elucidate the effects of Cd pollution stress on this process and the underline mechanisms. (3) to clarify the interaction and roles of Sphingobacterium sp. and earthworms in these processes. 4. Discussion. 1) There are seven subsections in this part, but the discussion seemed weak and not focused. In addition, since the authors present the "Discussion" part with the same order to that of the "Results" part, it is recommended that the authors combine them together and present them in the "Results and discussion" part. Otherwise, the authors need to make great effort to rewrite the "Discussion" part, in which the roles of earthworms, Sphingobacterium sp., and their combination should be discussed in a step-by-step and logical manner. 2) In the part of Effects of earthworms and Sphingobacterium sp. on soil carbon and nitrogen fractions, the authors should discuss the soil carbon and nitrogen cycling via experimental results and previous studies. Answer: Thanks for your comment and suggestion. You are right. This part existed serious weaknesses in discussion and lack of focus. After discussion, we rewrite and improve the "Discussion" part and also discuss our result with previous studies as: Lin 293-417: 4.1 The effects of Sphingobacterium sp. on C mineralization Microorganisms are known to contribute to the mineralization and loss of SOC [START_REF] Schimel | Microbial control over carbon cycling in soil[END_REF]. Sphingobacterium sp. is a type of gram-negative bacteria. Likewise, previous studies [START_REF] Shankar | Saccharification of the pretreated 592 husks of corn, peanut and coffee cherry by the lignocellulolytic enzymes secreted by 127[END_REF][46] used Sphingobacterium sp. to decompose the agricultural byproducts and to biodegrade organic pollutants. The performance of Sphingobacterium sp. in both noncontaminated and Cd contaminated soils indicates its ability to mineralize carbon and tolerate metal stress. These abilities align with a similar study [47], who demonstrated that the inoculation of Sphingobacterium sp. can enhance the degradation of organic pollutant residues in Cd (0.30 mg•kg-1) contaminated soil. It also can be inferred from the aforementioned results that these functions of Sphingobacterium sp. were not dependent on the gut conditions of earthworms. Additionally, the highest microbial biomass carbon content and the microbial quotient carbon in the CS treatment (Fig. 1C and Fig. 2E) were due to the inoculation of this strain, its adaption in the soil environment and substantial proliferation. However, this strong adaptive phenomenon was not observed in Cd-contaminated soil, where there is reduced microbial biomass carbon caused by metal stress. For instance, the adaptation process of Sphingobacterium sp. was observed in the early stage (60 days) in Cd-contaminated soil (0.3 mg•kg-1), with the strain population size gradually increasing after 90 days [47]. Nevertheless, this study only lasted for 20 days and more time is needed for Sphingobacterium sp. to complete the adaption processes under metal stress. 4.2 Earthworm survival, growth and species-specific effects on C and N transformation Earthworm abundance and biomass decreased after 20 days of cultivation in both soils, especially in Cd-contaminated soil, consistent with findings from a previous study [29]. This decline may be attributed to soil nutrient limitation and the toxic effects of high Cd concentration on earthworms [48]. Regarding earthworm species, A. gracilis showed higher resistance to environmental stresses than E. fetida, probably owing to the different strategies employed by earthworms in metal handling and resistance. In a previous study, a toxicokinetic model was employed, which found that wild earthworm species were more specialized than E. fetida in balancing the trade-off between metal uptake and excretion processes based on their surrounding environment [49]. There are different effects of earthworms on soil C and N transformation in noncontaminated and Cd-contaminated soil. In non-contaminated soil with a high organic carbon content, earthworms alone facilitated soil organic carbon and nitrogen mineralization. This is consistent with the trend of carbon and nitrogen changes observed in vermicomposting [50], presumably through the decomposition of organic matter by specific microorganisms under the unique conditions within the earthworm gut [51]. In addition, it can be found that earthworm activities increased the soil microbial quotient carbon compared to the control, indicating improved efficiency of soil organic matter utilization by microorganisms [52]. However, soil carbon stabilization in Cd-contaminated soil was probably due to earthworms consuming more organic matter to resist metal toxicity [START_REF] Ge | Eisenia fetida impact on cadmium 620 availability and distribution in specific components of the earthworm drilosphere[END_REF]. Moreover, it has been proved that gut peristalsis associates partly degraded organic matter with minerals [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF] and leads to formation of organic matter-rich aggregates [START_REF] Hong | How do 627 earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Guhra | Earthworm mucus contributes to the formation 630 of organo-mineral associations in soil[END_REF], which are conducive to SOC sequestration [7,[START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Nannoni | Soil properties and metal accumulation by earthworms in the 637 Siena urban area (Italy)[END_REF][START_REF] Nyenda | Changes in physicochemical properties on a 672 chronosequence of gold mine tailings[END_REF][60]. It was concluded that earthworms can slow down SOC mineralization in the presence of more minerals [52]. Therefore, earthworm activities reduced carbon mineralization in mineral-rich Cd-contaminated soil. Furthermore, this effect may also be explained by the fact that contaminated soils contain stable carbon that is not easily mineralized. The positive correlation between β-glucosidase and N-acetyl-β-D-glucosaminidase activity, demonstrated with PCA, also supports the notion that soil microorganisms tend to use the active carbon pool generated by the decomposition of humus formed by litter (Fig. 5A; Fig. S1), and this favors the accumulation of recalcitrant soil carbon pools. The earthworm species have great impact on biogeochemical cycling through their feeding habits and cast properties [31]. In non-contaminated and Cd-contaminated soil, both E. fetida and A. gracilis perform silimar ecological functions; however, their performance is also influenced by earthworm survival strategies. In environments without metal stress, both species can enhance C and N mineralization. It is worth noting that E. fetida has a more pronounced effect on soil carbon and nitrogen mineralization than A. gracilis does (Fig. 1B andE; Fig. 2A andC). This could be attributed to the preference of E. fetida for ingesting high-quality organic matter. Their castings have a high level of particulate organic matter content and porosity that provides plentiful food and spaces for soil microorganisms [31], thereby accelerating the decomposition of organic matter [61][62][START_REF] Yadav | Role of earthworms in soil health and variablesinfluencing their 652 population dynamic: a review[END_REF]. On the contrary, the wild earthworm A. gracilis not only fed on soil organic matter, but also on more mineral particles. In addition, E. fetida found it easier to obtain food in non-contaminated soil with a relatively high organic matter content (Table 1). A high carbon to nitrogen ratio was favored for earthworm growth [START_REF] Fusilero | Weed management 654 systems and other factors affecting the earthworm population in a banana plantation[END_REF], so this species performed better. In contrast, in Cdcontaminated soil, the mineralization processes of soil organic matter slow down. The wild earthworm species A. gracilis demonstrated better survival and had more significant effects on C stabilization than E. fetida (Table 2, Fig. 1 and Fig. 2). The low organic matter content in Cd-contaminated soils limited the decomposition functions of E. fetida. Furthermore, earthworms need to consume more food to resistant Cd stress. Both species increases their food consumption to feed on more organic matter. In this process, earthworms were compelled to ingest more mineral particles in soils characterized by low organic matter (Table 1) and high soil mineral content. The gutgrazing process facilitated the thorough mixing and binding of organic matter with soil minerals [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF]. As for enzymes, the enzymes activity was influenced by earthworm species and metal stress. Likewise, in the previous study, earthworm activities suppressed β-glucosidase activity during the stimulation of C mineralization processes [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF]. It was speculated that when soil microorganisms could easily access the readily Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation mineralized C released by earthworms, they would use a smaller portion of the carbon from enzyme-catalyzed decomposition of soil organic carbon. Enzyme activity only represents the concomitant consequence of earthworm activities but does not serve as the major driver of organic matter decomposition. It is also controlled by multiple factors such as food type, pH, temperature, gut transit time, metal stress, substrates, et al., [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF][START_REF] Karasov | Ecological physiology of diet and digestive 661 systems[END_REF][START_REF] Utobo | Soil enzymes as bioindicators of soil ecosystem status[END_REF].

The combined effects of earthworm and Sphingobacterium sp. on the balance between C and N stabilization and mineralization

The combination processes showed that Sphingobacterium sp. assisted earthworms in further promoting carbon and nitrogen mineralization in noncontaminated soils (Fig. 4A andC) and carbon stabilization in Cd-contaminated soils (Fig. 5A andC), as reflected by the increased contents of mineralization (DOC, AN) and the stabilization indicators (SOC), respectively. It was also found that inoculated earthworm can process organically polluted water and excess sludge, and Sphingobacterium, known for its high ability to degrade organic matter, is one of the most abundant microbes in earthworm castings [START_REF] Liu | Effect of earthworms on the performance 666 and microbial communities of excess sludge treatment process in vermifilter[END_REF]. Nevertheless, our study demonstrated that external addition of Sphingobacterium sp. can help earthworms adapt more easily to environment and exert their ecological functions. Despite having similar effects when used in combination or when being inoculated alone, the strategies of the two species differed in non-contaminated and Cd-contaminated soil (Fig. 4D). Compared with treatments that inoculate earthworms alone, CSA and CSE treatments further activated INV and PRO activities in non-contaminated soil (Fig. 3 and Fig. 4D); On the other hand, SSA further activated INV activities and inhibited PRO activities, while SSE further inhibited URE activities (Fig. 3 and Fig. 5D). It is well known that soil enzyme activity is mainly derived from microorganisms and can reflect microbial activity [START_REF] Li | Microbial biomass, 669 enzyme and mineralization activity in relation to soil organic C, N and P turnover influenced by 670 acid metal stress[END_REF]. Inoculation of Sphingobacterium sp. may stimulate microbial activities in the soil and the earthworm gut in non-contaminated soil [16]. In Cdcontaminated soil, it may represent the natural response of earthworms to the inoculation of Sphingobacterium sp. and metal stress. Compared to non-contaminated soil, metal stress induced inhibition of PRO activities in the treatment with A. gracilis inoculation and URE activities in the treatment with E. fetida inoculation, indirectly demonstrating distinct coping strategies for metal stress among earthworm species. Given that researchers have [START_REF] Akinnuoye-Adelabu | Effect of 673 redworm extracts against Fusarium root rot during wheat seedling emergence[END_REF] found that the vermiwash and mucus extracted from earthworms can greatly inhibit the growth of pathogenic fungi, it is also possible that the mucus compounds changed due to metal pollutant stress in this study, such as secretion of specific organic substances to protect itself from physiological damage, while inhibiting the microbial activities. The underlying mechanisms are still not clear and require further research. In these processes, earthworm activities dominated the soil carbon and nitrogen mineralization processes, while Sphingobacterium sp. played a supporting role in combined processes. When the effects of earthworm and Sphingobacterium sp. on carbon and nitrogen were consistent, they enhanced carbon mineralization; otherwise they undermined this process. Hence, in non-contaminated soil, carbon mineralization was observed. While in Cd-contaminated soil, carbon storage was more prevalent due to the stronger effect of earthworms on stabilization than Sphingobacterium sp. on mineralization. This is further confirmed by PCA which demonstrated that earthworms play a critical role in soil organic carbon decomposition processes, following the same trend as inoculation with earthworm alone and in combination with Sphingobacterium sp. (Fig. 4A andC; Fig. 5A andC). Additionally, in this system, a strong interaction existed between earthworms and Sphingobacterium sp.. Earthworm activities under the combined process dramatically decreased microbial biomass carbon compared to Sphingobacterium sp. activities alone. It is suggested that earthworms contribute to maintaining the ecosystem balance and stabilizing the microbial community structure through feeding [START_REF] Zhang | Influence of the earthworm Pheretima guillelmi on soil 676 microbial biomass and activity[END_REF] and regulating the allocation of active organic carbon and dissolved organic carbon pools [START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Yu | Effects of straw application and earthworm inoculation on 679 soil labile organic carbon[END_REF]. Therefore, the rapid proliferation of Sphingobacterium sp. was prevented in CSE and CAS treatments. It was further confirmed by the significantly lower microbial quotient carbon in treatment CSE and CSA compared to treatment CS (Fig. 2E). It can be deduced that earthworms may reduce the conversion efficiency of soil microorganisms in utilizing carbon sources into microbial biomass carbon [52] or they can intensely feed and graze on microbes [10,[START_REF] Vafa | Sewage sludge application strongly modifies earthworm 681 impact on microbial and biochemical attributes in a semi-arid calcareous soil from Iran[END_REF][START_REF] Edwards | Interactions between earthworms, microorganisms, and 684 other invertebrates[END_REF][START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF]. Additionally, the different impact of earthworm species may be caused by E. fetida being more conducive to the survival and growth of G-bacterial communities [START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF], thus promoting Sphingobacterium sp. to assist earthworms in carbon mineralization. Based on the above, another underlying factor was postulated that

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Sphingobacterium sp. may assist earthworms in mineralizing soil organic carbon and nitrogen by stimulating the microbial activities in the earthworm gut. The subsequent studies need to focus on the earthworm gut microbial to reveal the internal mechanisms. 5. Writing and grammar. There are a number of sentences that need improvement. For example, 1) The clean soil in this study means non-Cd-contaminated soil. Therefore, it should be better to use the Cd-contaminated and non-contaminated soils to describe these two soils. Answer: Thanks for your comment and suggestion. We agree with you and have been replaced "clean soil and contaminated soil" to "non-contaminated soil and Cdcontaminated soil" to describe these two soils.

2) INV, inverse activity (in the figure caption of Fig. 3) has a spelling error. Answer: Thanks for your comment. This word in the full text has been revised.

3) The name of earthworm in this study should be "E. fetida" and "A. gracilis", not "E.fetida" and "A.gracilis". Answer: Thanks for your comment. Sorry about this mistake. The abbreviation format of earthworm species in the full-text has been revised as "E. fetida" and "A. gracilis". 4) There should be a space before the parentheses and brackets. Many similar typing mistakes can be found through the manuscript. Please carefully check and revise them. Answer: Thanks for your reminding and suggestion. The full-text has been checked and revised. 6. Please find a professional editing service or a native speaker of English to check the language. Answer: Thanks for your suggestion. This manuscript of new version has been checked and polished by the professional institute. 7. Data presentation. 1) The text in the figures was too small to match the text in the manuscript. Try not to abbreviate the titles of vertical coordinates. Answer: Thanks for your comment and suggestion. The text of all the figures was enlarged. We replaced the abbreviation by the full name in the titles of vertical coordinates. In fig. 2, there do not have more space to put the full name of the ratio such as DOC:DON, C:N, AN:TN, and MBC:MBN. Therefore, we keep same vertical coordinates with before and noted it under the figure. Thanks for your understanding. Like below: Fig. 1. Soil carbon and nitrogen form characteristics. Results are expressed as the mean value ± SE (n = 3). Different lowercase letters indicate significantly differences (P < 0.05) among the treatments. Fig. 2. The ratio of soil carbon and nitrogen forms. SOC, soil organic carbon; DOC, dissolved organic matter; TN, total nitrogen; AN, available nitrogen; C:N, soil organic carbon-to-total nitrogen ratio; MBC, microbial biomass carbon; MBN, microbial biomass nitrogen. Results are expressed as the mean value ± SE (n = 3). Different lowercase letters indicate significantly differences (P < 0.05) among the treatments. Fig. 3. Characterization of soil enzyme activity. Results are expressed as the mean value ± SE (n = 3). Different lowercase letters indicate significantly differences (P < 0.05) among the treatments.

2) Table 2. The significance difference in biomass on the 20th day (a, b) was mislabeled. Answer: Thanks for your suggestion. We revised the data format and check the significance between the treatments, like below: Table2 Survival rate and mean biomass loss of earthworm at the 20th day TreatmentsSurvival rate (%)Mean biomass loss (%) CE95.2 ± 4.76a17.4 ± 13.3b CA86.1 ± 7.35a7.68 ± 4.54b CSE91.1 ± 4.49a19.4 ± 5.26b CSA87.8 ± 6.19a13.7 ± 10.2b SE77.8 ± 11.1a17.6 ± 5.22b SA100 ± 0a18.9 ± 5.54b SSE31.2 ± 6.10b77.2 ± 3.99a SSA100 ± 0a18.9 ± 3.27b Note: Results are expressed as the mean value ± SE (n = 3). The initial letters C and S represents the soil I and soil II. CE, CA, CSE and CSA represents the treatments with E. fetida, A. gracilis, Sphingobacterium sp. + E.fetida and Sphingobacterium sp. + A.gracilis in soil I, while SE, SA, SSE and SSA represents the treatments with E. fetida, A. gracilis, Sphingobacterium sp.+ E. fetida and Sphingobacterium sp. + A. gracilis in soil II, respectively. Different lowercase letters indicate significant differences (P < 0.05) between different treatments. 8. Line 81-82. "Earthworm selective feeding activities have a significant impact on soil carbon and nitrogen cycling in gut [6]." What do you mean "soil carbon and nitrogen cycling in gut"? Please clarify. Answer: Thanks for your comment. Sorry for our omission. It should be earthworm selective feeding activities have a significant impact on soil carbon and nitrogen cycling. We have revised as: Line 89-90: Earthworm's preferential feeding and concentration processes have a substantial impact soil carbon and nitrogen cycling [7]. 9. Line 138-139. Why did the authors select two epigeic species of earthworms in this study? Please clarify. Answer: Thanks for your comment. The different species and ecological categories have different impacts on soil organic matter decomposition and soil C and N cycles. The species of the experimental model earthworm E. fetida and local wild epigeic species may have different survival strategies that affect microbial biomass or activities, resulting in their different performances in soil carbon and nitrogen cycles in non-contaminated and Cd-contaminated soil. Importantly, it is worth to be noticed that the different effects of them and trying to convert laboratory tests into field experiments. We clarified in the "Introduction" part as: Line 112-117: In addition, different earthworm species and ecological categories have varying effects on soil organic matter degradation and soil C and N cycles [31]. The species of the experimental model earthworm E. fetida and local wild epigeic species may have different survival strategies that affect microbial biomass or activities, resulting in their different performances in soil carbon and nitrogen cycles in noncontaminated and Cd-contaminated soil. 10. Line 139. The abbreviation of Amynthas gracilis is in a wrong format. A space should be added between the genus name and the species name. Many similar typing mistakes can be found through the manuscript. Please carefully check and revise them. Answer: Thanks for your comment. The abbreviation format of earthworm species has been revised as "E. fetida" and "A. gracilis". In addition, the full-text has been checked and revised. 11. Line 144-146. What is microbial CM0827 Propionibacterium sp. medium? Please clarify why use this kind of medium. In addition, there should be no commas followed by consist of. Answer: Thanks for your comment and suggestion. The CM0827 Propionibacterium sp. medium means medium with the number CM0827 and the name Propionibacterium sp. medium, which can support the growth of gram-negative bacteria. Thanks for your kind reminding. We have deleted the punctuation followed by consist of. 12. Line 147. The pH and OD405 value 1.45 should be placed after the recipe of medium and before the incubation of bacteria in the tested soils, respectively. By the way, what do you mean "set aside"? Please clarify. Answer: Thanks for your comment and suggestion. Sorry to describe not clear. We already modified it as: Line 165-168: CM0827 Propionibacterium sp. medium, which consisted of tryptone 15 g, L-cystine 0.5 g, yeast extract 5 g, sodium thioglycolate 0.5 g, glucose 5 g, agar 0.7 g, sodium chloride 2.5 g, resazurin 0.001 g, distilled water 1 L, was employed to cultural Sphingobacterium sp. and was set at pH 7.0. In addition, the means of "set aside" is "prepared the experiment materials waiting to be used in the subsequent experiments". We already revised it as: Line 168-169: The strain was incubated at a constant temperature of 30°C for 2 days before inoculation into the soil. 13. Line 180-182. This sentence needs revision. For example, "Total cadmium (Cd) content" of whom? What was the certified standard material? Please provide. Answer: Thanks for your comment. We added the explanation of DTPA, the determination methods of DTPA and total Cd content of soil, and the certified standard materials. This part be revised as: Line 203-213: 2.4 Total and DTPA extractable Cadmium content Total cadmium (Cd) in the soils was extracted by microwave digestion with aqua regia (HNO3:HCl (v/v) = 1:3). The diethylenetriaminepentaacetic acid (DTPA) extractable form was regarded as bioavailable metals and toxic. DTPA-extractable Cd was extracted with DTPA extractant at a Soil:DTPA extractant ratio of 5:25 (g/mL), which included a solution of 5 Mm DTPA and 10 mM CaCl2. The extracted suspensions were analyzed for total cadmium (Cd) and DTPA-extractable contents using inductively coupled plasma-mass spectrometry (ICP-MS, 7700ce, Agilent Technologies, Santa Clara, CA, USA) [43]. GBW07455 (GSS-26) was employed as certified reference material for soil analyses, and the measured concentrations were within 98% of the reported certified concentrations for Cd. Quality control measures included three repetitions for each soil and three blanks for each batch to ensure the quality of the analysis. 14. Line 205-207. This sentence is very confusing. Please rewrite it. Answer: Thanks for your comment. We revised the data format order to show the contents of Line 205-207 in Table 2, and deleted Line 205-207. It will be clearer and easier to understand than before. 15. Line 251-253. Technically, INV activity is one of the enzymes activity. Please revise this sentence. Answer: Thanks for your comment. This sentence really induced the misunderstanding. We deleted ", but greater variation in the effect on INV activities." in Line 252-253 after reconsidering. 16. Line 394-399. This sentence is too long and hard to digest. Please rewrite. Answer: Thanks for your comment. Because we reorganized the discussion part, the relevant contents were revised as: Line 327-328: However, soil carbon stabilization in Cd-contaminated soil was probably due to earthworms consuming more organic matter to resist metal toxicity [START_REF] Ge | Eisenia fetida impact on cadmium 620 availability and distribution in specific components of the earthworm drilosphere[END_REF]. Moreover, it has been proved that gut peristalsis associates partly degraded organic matter with minerals [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF] and leads to formation of organic matter-rich aggregates [START_REF] Hong | How do 627 earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Guhra | Earthworm mucus contributes to the formation 630 of organo-mineral associations in soil[END_REF], which are conducive to SOC sequestration [7,[START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Nannoni | Soil properties and metal accumulation by earthworms in the 637 Siena urban area (Italy)[END_REF][START_REF] Nyenda | Changes in physicochemical properties on a 672 chronosequence of gold mine tailings[END_REF][60]. It was concluded that earthworms can slow down SOC mineralization in the presence of more minerals [52]. Therefore, earthworm activities reduced carbon mineralization in mineral-rich Cdcontaminated soil.

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

Dear Editor, We would like to submit a manuscript entitled "Different combined effects of earthworm and Sphingobacterium sp. on soil C, N forms and enzyme activities in clean and Cd-contaminated soil" for possible publication in European Journal of Soil Biology.

Soil carbon and nitrogen dynamics is efficiency for climate change and environment resource. As "soil ecosystem engineers", earthworm have a significant impact on soil carbon and nitrogen cycling. In our previous studies, Sphingobacterium sp., a kind of gram-negative bacterium, was demonstrated as a kind of core microorganism in earthworm gut. To date, some previous studies evidenced that Sphingobacterium sp. has a strong degrading capacity for organic pollutants. Nevertheless, the relationship between Sphingobacterium sp. and the processes of earthworms affecting on soil organic matter degradation has not been studied. Moreover, the effect of Sphingobacterium sp. in combination with earthworms on the soil carbon and nitrogen cycle have not yet been investigated as well. The effect of metal contamination, which severely affects the environmental quality and organisms, on this process are not clear.

In our study, two epigeic species of earthworm (E.fetida and A.gracilis) and Sphingobacterium sp. were inoculated in clean and cadmium-contaminated soil. We investigated their single or combined effects on carbon and nitrogen forms, enzyme activities and evaluated their interaction on soil C and N cycles, investigating how Sphingobacterium sp. assist earthworm in influencing soil carbon stabilization and nitrogen cycling under clean and metal contaminated soil. The significance and findings are as follows: Earthworm promoted mineralization of C and N in clean soil and C storage in contaminated soil. Inoculation Sphingobacterium sp. individually facilitated soil carbon and nitrogen mineralization in both soils, and this effect was enhanced by combined with earthworms in clean soil. In addition, E.fetida was more effective than that of A.gracilis. While in cadmium (Cd) contaminated environment, soil exhibited carbon storage under the combination inoculated Sphingobacterium sp. with earthworm and no significant effects of earthworm species.

This study provides a clear understanding on the different effects of earthworm and Sphingobacterium sp. on soil carbon and nitrogen cycling in clean and contaminated soil.

We hereby certify that this paper consists of original, unpublished work which is not under consideration for publication elsewhere and all authors in agreement with the content of the manuscript have contributed significantly.

Thank you very much for your considering our manuscript for potential publication. We are looking forward to hearing from you soon.

Sincerely,

The manuscript has 40 pages including:

pages of text, pages of tables and figures page of supplementary figure

Responses to reviewers Dear Editors and Reviewers,

Thank you very much for your careful review of our paper. The feedback is very valuable. We have revised the manuscript in response to your suggestions and questions. All the modifications made according to reviewer's comments are highlighted in yellow in our manuscript. Our responses are also outlined below following your comments. I hope the revised manuscript is acceptable for publication in our journal. The manuscript aims to examine the roles of one of dominant gut bacteria taxa (Sphingobacterium sp.) of earthworms and two earthworm species on soil organic matter transformation, as well as the associated changes in soil enzyme activities and other soil properties. Two types of soil, clean and Cd-contaminated, were used, and the results were interesting. There are some suggestions to be carefully considered in the revision.

1. First, one or two scientific hypotheses could be given. In lines 91-92, the authors mentioned that Sphingobacterium sp. was usually one of dominant bacteria taxa in earthworm guts. This is very important base that a scientific hypothesis could be raised on. For example, given that Sphingobacterium sp. was dominant in worm gut, an inoculation of this bacteria may exert similar impacts on organic matter decomposition like earthworms, if it contributed great to the worm gut-mediated organic matter decomposition. or Sphingobacterium sp. inoculation did not affect organic matter transformation like earthworms, if its function relies on the special gut condition. or Sphingobacterium sp. may enhance earthworm impacts on carbon transformation by increasing the microbial activities in earthworm gut? Answer: Thanks for your comment and suggestion. Earthworm's preferential feeding and concentration processes have a substantial impact soil carbon and nitrogen cycling. Their bioturbation activities, including burrowing, feeding, and excretion (casts and surface mucus), affects the soil carbon and nitrogen distribution within soil profile. These effects result from complex mutualism interactions between earthworms and microorganisms. Previous studies have established connections between earthworms and rhizosphere growth-promoting bacteria or specific arbuscular mycorrhizal fungi to enhance nutrient availability or nitrogen storage in soil.

Moreover, the collaboration of indigenous earthworms and gut bacteria has been employed to remediate metal contaminated soil and has shown superiority compared to chemical amendments.

Likewise, combining earthworms with dominate gut bacteria taxa to enhance organic matter decomposition is also advantageous, because it is more readily accepted by earthworms. In our previous studies, Sphingobacterium sp. was identified as a dominant bacterial taxon in the earthworm gut. Sphingobacterium sp., a gram-negative bacterium, exhibits a wide range of nutrient sources, high adaptability, and a broad ecological distribution. In practice, it has been used to decompose agricultural waste and degrade organic pollutants (e.g. alkanes and polycyclic aromatic hydrocarbons) in soil. Therefore, this decomposition ability of earthworms probably derives from symbiotic microorganisms in their guts. Considering that earthworms and Sphingobacterium sp. impact organic matter decomposition and the latter was dominant bacterial taxon in earthworm gut, a synergistic relationship may exist, enhancing soil organic matter decomposition. However, under environment stress, such as metal contamination, the activities and complex interactions of them may be suppressed. Specifically, metal contamination can induce biomass loss, reduce survival rate, cause irreversible physiological damage, decrease the stability of gut bacterial network, and dampen its ecological functions. Similarly, the inhibition effects also impact soil microorganisms, leading to reduced soil enzyme functions critical for organic matter cycling. In addition, different earthworm species and ecological categories have varying effects on soil organic matter degradation and soil C and N cycles. The species of the experimental model earthworm E. fetida and local wild epigeic species may have different survival strategies that affect microbial biomass or activities, resulting in their different performances in soil carbon and nitrogen cycles in non-contaminated and Cd-contaminated soil.

Based on this, the same taxa were selected in this study to reveal that if the combined effects of this strain and earthworm on soil carbon and nitrogen turnover. Therefore, we added the hypothesis as: Line 135-140: Given that Sphingobacterium sp. is the dominant bacterial taxon in the earthworm gut, we hypothesized that Sphingobacterium sp. and earthworm could individually promote soil organic matter decomposition and, when combined, exert reinforcing effects. These effects are negatively impacted by metal stress and influenced by the survival strategies of different earthworm species; Additionally, Sphingobacterium sp. and earthworms may play different roles in this combined process, with Sphingobacterium sp. stimulating microbial activities in the earthworm gut.

2. In addition, the possible interactions (and the associated consequences) between Sphingobacterium sp. and earthworms may differ in the clean and Cd-contaminated soils. Q: What may be the difference and how did they happen? All these should be added in the introduction section and reflected in the hypotheses. For instance, in considering what..., earthworm and Sphingobacterium sp. will enhance soil carbon mineralization in clean soil, but enhance soil carbon sequestration in Cd-contaminated soil..; or, given that ...., the inoculated earthworm and bacteria may cooperate and exert an additive effect, or may compete or negatively interact with each other, and exert an negative effect? Answer: Thanks for your comment. We rearrange the introduction part as: Line 108-117: However, under environment stress, such as metal contamination, the activities and complex interactions of them may be suppressed. Specifically, metal contamination can induce biomass loss, reduce survival rate, cause irreversible physiological damage, decrease the stability of gut bacterial network, and dampen its ecological functions [27][28][29]. Similarly, the inhibition effects also impact soil microorganisms, leading to reduced soil enzyme functions critical for organic matter cycling [30]. In addition, different earthworm species and ecological categories have varying effects on soil organic matter degradation and soil C and N cycles [31]. The species of the experimental model earthworm E. fetida and local wild epigeic species may have different survival strategies that affect microbial biomass or activities, resulting in their different performances in soil carbon and nitrogen cycles in non-contaminated and Cd-contaminated soil.

In addition, we also added the hypothesis as: Line 135-140: Given that Sphingobacterium sp. was dominant bacteria taxa in earthworm gut, we hypothesized that Sphingobacterium sp. and earthworm could promote soil organic matter decomposition in individual processes and exert reinforcement effects in combined processes, which effects are negatively impacted by metal stress and influenced by survival strategies of different earthworm species; Additionally, Sphingobacterium sp. and earthworms may play different roles in combined process in this system, with Sphingobacterium sp. making influence by stimulating microbial activities in earthworm gut.

3. Line 132, please check the biomass per ind. here, too big for a E. fetida.

Answer: Thanks for your comment. We added more detail about earthworm biomass after checking and confirming the data as "Each pot was inoculated with 3 g of earthworms, Answer: Thanks for your comment. We added more details about DTPA and measure method in lines 203-213 as:

Total and DTPA extractable Cadmium content

Total cadmium (Cd) in the soils was extracted by microwave digestion with aqua regia (HNO3:HCl (v/v) = 1:3). The diethylenetriaminepentaacetic acid (DTPA) extractable form was regarded as bioavailable metals and toxic. DTPA-extractable Cd was extracted with DTPA extractant at a Soil:DTPA extractant ratio of 5:25 (g/mL), which included a solution of 5 Mm DTPA and 10 mM CaCl2. The extracted suspensions were analyzed for total cadmium (Cd) and DTPA-extractable contents using inductively coupled plasma-mass spectrometry (ICP-MS, 7700ce, Agilent Technologies, Santa Clara, CA, USA) [43]. GBW07455 (GSS-26) was employed as certified reference material for soil analyses, and the measured concentrations were within 98% of the reported certified concentrations for Cd. Quality control measures included three repetitions for each soil and three blanks for each batch to ensure the quality of the analysis. 5. Lines 322-326, Not sure here, a lower standing MBC did not necessarily indicate a low growth of bacterial population, alternatively, this may also result from a strong feeding of earthworm on the bacterial biomass? Answer: Thanks for your comment. We agree with you. The feeding of earthworm also strong impacts the microbial biomass and structure. We revised and improve this part as: Line 402-411: Earthworm activities under the combined process dramatically decreased microbial biomass carbon compared to Sphingobacterium sp. activities alone. It is suggested that earthworms contribute to maintaining the ecosystem balance and stabilizing the microbial community structure through feeding [START_REF] Zhang | Influence of the earthworm Pheretima guillelmi on soil 676 microbial biomass and activity[END_REF] and regulating the allocation of active organic carbon and dissolved organic carbon pools [START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Yu | Effects of straw application and earthworm inoculation on 679 soil labile organic carbon[END_REF]. Therefore, the rapid proliferation of Sphingobacterium sp. was prevented in CSE and CAS treatments. It was further confirmed by the significantly lower microbial quotient carbon in treatment CSE and CSA compared to treatment CS (Fig. 2E). It can be deduced that earthworms may reduce the conversion efficiency of soil microorganisms in utilizing carbon sources into microbial biomass carbon [52] or they can intensely feed and graze on microbes [10,[START_REF] Vafa | Sewage sludge application strongly modifies earthworm 681 impact on microbial and biochemical attributes in a semi-arid calcareous soil from Iran[END_REF][START_REF] Edwards | Interactions between earthworms, microorganisms, and 684 other invertebrates[END_REF][START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF].

6.

Lines 355-356 and lines 414-415, so, how to understand such negative effects of earthworms? similar negative effect was also reported in literature, such as Lv et al., 2020 https://doi.org/10.1016/j.scitotenv.2020.137227; Answer: Thanks for your comment. Likewise, in [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF], earthworm activities suppressed β-glucosidase activity during stimulating carbon mineralization processes. They speculated that when soil microorganisms could easily access the readily mineralized C released by earthworms, they would use a smaller portion of the carbon from enzyme-catalyzed decomposition of soil organic carbon. Additionally, the enzyme activity only represents the concomitant consequence of earthworm activities but does not serve as the major driver of organic matter decomposition. It is also controlled by multiple factors such as food type, pH, temperature, gut transit time, metal stress, substrates, et al., [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF][START_REF] Karasov | Ecological physiology of diet and digestive 661 systems[END_REF][START_REF] Utobo | Soil enzymes as bioindicators of soil ecosystem status[END_REF].

We revised the discussion of negative effects as: Line 358-365 as: As for enzymes, the enzymes activity was influenced by earthworm species and metal stress. Likewise, in the previous study, earthworm activities suppressed β-glucosidase activity during the stimulation of C mineralization processes [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF]. It was speculated that when soil microorganisms could easily access the readily mineralized C released by earthworms, they would use a smaller portion of the carbon from enzyme-catalyzed decomposition of soil organic carbon.

Enzyme activity only represents the concomitant consequence of earthworm activities but does not serve as the major driver of organic matter decomposition. It is also controlled by multiple factors such as food type, pH, temperature, gut transit time, metal stress, substrates, et al., [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF][START_REF] Karasov | Ecological physiology of diet and digestive 661 systems[END_REF][START_REF] Utobo | Soil enzymes as bioindicators of soil ecosystem status[END_REF].

7. lines 414-415 may suggest that soil microbiota did not play a key role in OC decomposition processes, but changed accordingly, worm dominated these processes.

Answer: Thanks for your comment. You are right. The soil enzymes activities shown inhibited which may suggest that the earthworm dominated the organic carbon decomposition processes and Sphingobacterium sp. play an assistance role. Another underlying factor is that Sphingobacterium sp. may assist earthworms in mineralizing soil organic carbon and nitrogen by stimulating the microbial activities in the earthworm gut. The subsequent studies need to focus on the earthworm gut microbial to reveal the internal mechanisms. We have added some discussion in the manuscript as: Line 393-417: In these processes, earthworm activities dominated the soil carbon and nitrogen mineralization processes, while Sphingobacterium sp. played a supporting role in combined processes. When the effects of earthworm and Sphingobacterium sp. on carbon and nitrogen were consistent, they enhanced each other; otherwise they undermined each other. Hence, in non-contaminated soil, carbon mineralization was observed. While in Cd-contaminated soil, carbon storage was more prevalent due to the stronger effect of earthworms on stabilization than Sphingobacterium sp. on mineralization. This is further confirmed by PCA which demonstrated that earthworms play a critical role in soil organic carbon decomposition processes, following the same trend as inoculation with earthworm alone and in combination with Sphingobacterium sp.

(Fig. 4A andC; Fig. 5A andC). Additionally, in this system, a strong interaction existed between earthworms and Sphingobacterium sp.. Earthworm activities under the combined process dramatically decreased microbial biomass carbon compared to Sphingobacterium sp. activities alone. It is suggested that earthworms contribute to maintaining the ecosystem balance and stabilizing the microbial community structure through feeding [START_REF] Zhang | Influence of the earthworm Pheretima guillelmi on soil 676 microbial biomass and activity[END_REF] and regulating the allocation of active organic carbon and dissolved organic carbon pools [START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Yu | Effects of straw application and earthworm inoculation on 679 soil labile organic carbon[END_REF]. Therefore, the rapid proliferation of Sphingobacterium sp. was prevented in CSE and CAS treatments. It was further confirmed by the significantly lower microbial quotient carbon in treatment CSE and CSA compared to treatment CS (Fig. 2E). It can be deduced that earthworms may reduce the conversion efficiency of soil microorganisms in utilizing carbon sources into microbial biomass carbon [52] or they can intensely feed and graze on microbes [10,[START_REF] Vafa | Sewage sludge application strongly modifies earthworm 681 impact on microbial and biochemical attributes in a semi-arid calcareous soil from Iran[END_REF][START_REF] Edwards | Interactions between earthworms, microorganisms, and 684 other invertebrates[END_REF][START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF]. Additionally, the different impact of earthworm species may be caused by E. fetida being more conducive to the survival and growth of G -bacterial communities [START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF], thus promoting Sphingobacterium sp. to assist earthworms in carbon mineralization. Based on the above, another underlying factor was postulated that Sphingobacterium sp. may assist earthworms in mineralizing soil organic carbon and nitrogen by stimulating the microbial activities in the earthworm gut. The subsequent studies need to focus on the earthworm gut microbial to reveal the internal mechanisms.

8. lines 392-393, how could the assistance role of Sphingobacterium sp. on worm could be observed in Fig. 4a? Answer: Thanks for your comment. Sorry for our mistakes. This conclusion cann't be observed only in Fig. 4A, it should be analysis through combining Fig. 4A Line 399-401: This is further confirmed by PCA which demonstrated that earthworms play a critical role in soil organic carbon decomposition processes, following the same trend as inoculation with earthworm alone and in combination with Sphingobacterium sp. (Fig. 4A andC; Fig. 5A andC).

Reviewer #2:

Jia et al. investigated the effect of single and combined application of earthworms and Sphingobacterium sp. on the C and N cycles in a non-contaminated soil and a cadmium-contaminated soil. The C and N fractions in soil, ratio of C/N fractions, soil enzyme activities, relationships among the above parameters were determined. The results of this study can provide a better understanding of how different species of earthworms and their co-application with a key bacterium from the gut of earthworm regulate the carbon and nitrogen mineralization/fixation in the non-contaminated and Cd-contaminated acid soils. Yet, the current version of the manuscript still needs further modification and improvement, particularly the "Discussion" part. More details need to be addressed clearly as well.

1. Abstract. The abstract should be revised and clearly introduce which kind of results comes to which kind of key finding, not just simply mention the results.

Answer: Thanks for your suggestion. We added the key finding and revised the "abstract" part as: Line 35-75: Earthworms and Sphingobacterium sp. are known for their strong organic compound decomposition ability and wide distribution in soil. However, interactions of soil organic matter decomposition with soil properties and whether microbial species such as Sphingobacterium sp. could assist earthworms in carbon and nitrogen transformation in soil remain poorly understood. Earthworms (Eisenia fetida, Amynthas gracilis) and Sphingobacterium sp. were introduced in non-contaminated and cadmium-contaminated soils under controlled laboratory conditions for 20 days. We examined their individual or combined effects on carbon and nitrogen forms and related enzyme activities to assess their influence on soil C and N cycling.

Individual Sphingobacterium sp. inoculation led to significantly decreased organic carbon (SOC) contents, reducing it by 16.5% in non-contaminated soil and by 3.77%, in Cd-contaminated soil. It resulted in an increased microbial biomass carbon (MBC) contents, reaching 1685 ± 292 mg•kg -1 in non-contaminated soil. Individual introductions of E. fetida and A. gracilis caused a decline in SOC content in non-contaminated soil, but increased significantly dissolved organic carbon (DOC) and alkali-hydrolysable nitrogen (AN) contents by 75.8%, 53.6% and 32.9%, 20.9%, respectively.

In contrast, in Cd-contaminated soil, only the significant combined effects of earthworms and Sphingobacterium sp. were linked to significant increase in SOC contents, raising by 7.22% and 9.64% in E. fetida + Sphingobacterium sp. and A. gracilis + Sphingobacterium sp. treatments, respectively. In non-contaminate soil, the combined effects of earthworm and Sphingobacterium sp. further increased DOC and AN content by 212%, 134% and 31.3%, 25.4% in the treatments of E. fetida + Sphingobacterium sp. and A. gracilis + Sphingobacterium sp., respectively; the highest ratios of DOC to SOC and AN to total Nitrogen (TN) were found in the earthworm+Sphingobacterium sp. treatments as well. In non-contaminated soil, Sphingobacterium sp. and earthworms mainly influenced β-glucosidase (BG), urease (URE), N-acetyl-β-D-glucosaminidase (NAG) activities and fluorescein diacetate hydrolysis (FDA) hydrolysis, while in Cd-contaminated soil, they mainly influenced invertase (INV), NAG, URE, and protease (PRO) activities. Principal component analysis indicated that in non-contaminated soil, the earthworm activities dominated the mineralization processes of soil carbon and nitrogen, and Sphingobacterium sp. can intensify this process when it was inoculated in soil along with earthworms. Furthermore, both earthworm species increased C and N levels by elevated INV and PRO activities in combined inoculation. However, in contaminated soil, the impact of earthworm inoculation on soil C stabilization showed a species dependent pattern. E. fetida reduced C mineralization by decreasing URE activities, while A. gracilis enhanced C stabilization by increasing INV activities and decreasing PRO activities. In conclusion, earthworms played a key role in enhancing C and N mineralization in non-contaminated soil and promoting C stabilization in contaminated soil. Both earthworm species followed similar strategies in the former process but adopted different strategies in the latter. When introduced individually, Sphingobacterium sp. was able to promote mineralization in both soils, primarily assisting earthworms in improving carbon and nitrogen mineralization in non-contaminated soil but hindering these processes in Cd-contaminated soil. These findings provide insights into the combined effects of earthworms and microorganisms on carbon and nitrogen cycling.

Highlights.

1) In the second bullet of highlights, the full names of the earthworms should be provided.

Answer: Thanks for your reminding. We give the full names of the earthworms in the highlights.

2) The third bullet of highlights is very confusing. If the bacteria could not assist earthworm to improve C and N mineralization in the Cd-contaminated soil, please just delete "rather than contaminated soil".

Answer: Thanks for your comment and suggestion. We agree with you and delete "rather than contaminated soil". we revised it as "Sphingobacterium sp. could assist earthworms in improving C and N mineralization in non-contaminated soil.".

3) How is the effect of Sphingobacterium sp. itself? Please provide one more bullet of highlight for this.

Answer: Thanks for your comment and suggestion. We added the effects of inoculation with Sphingobacterium sp. alone. Like below: "Sphingobacterium sp. could promote C mineralization in both soils.".

Introduction.

1) What is the hypothesis for the potential effect of Sphingobacterium sp. on carbon and nitrogen cycling process in Cd-contaminated soil? The authors need to add more background information to support their hypothesis in the "Introduction" part.

Answer: Thanks for your comment. We rearrange the introduction part and supply the background information and the potential effect of invocation Sphingobacterium sp. alone as: Line 108-112: However, under environment stress, such as metal contamination, the activities and complex interactions of them may be suppressed. Specifically, metal contamination can induce biomass loss, reduce survival rate, cause irreversible physiological damage, decrease the stability of gut bacterial network, and dampen its ecological functions [27][28][29]. Similarly, the inhibition effects also impact soil microorganisms, leading to reduced soil enzyme functions critical for organic matter cycling [30].

We also added the hypothesis in as: Line135-140: Given that Sphingobacterium sp. is the dominant bacterial taxon in the earthworm gut, we hypothesized that Sphingobacterium sp. and earthworm could individually promote soil organic matter decomposition and, when combined, exert reinforcing effects. These effects are negatively impacted by metal stress and influenced by the survival strategies of different earthworm species; Additionally, Sphingobacterium sp. and earthworms may play different roles in this combined process, with Sphingobacterium sp. stimulating microbial activities in the earthworm gut.

2) The specific objectives were not consistent with the "abstract" and "discussion".

Answer: Thanks for your comment. We revised the specific objectives as: Line 143-148: The specific research objectives were (1) to analyze and compare the effects of Sphingobacterium sp. and the two earthworm species, when inoculated alone and in combination, on the carbon and nitrogen forms and related enzyme activities in non-and Cd-contaminated soils.

(2) to elucidate the effects of Cd pollution stress on this process and the underline mechanisms. (3) to clarify the interaction and roles of Sphingobacterium sp. and earthworms in these processes.

Discussion.

1) There are seven subsections in this part, but the discussion seemed weak and not focused. In addition, since the authors present the "Discussion" part with the same order to that of the "Results" part, it is recommended that the authors combine them together and present them in the "Results and discussion" part. Otherwise, the authors need to make great effort to rewrite the "Discussion" part, in which the roles of earthworms, Sphingobacterium sp., and their combination should be discussed in a step-by-step and logical manner. 2) In the part of Effects of earthworms and Sphingobacterium sp. on soil carbon and nitrogen fractions, the authors should discuss the soil carbon and nitrogen cycling via experimental results and previous studies.

Answer: Thanks for your comment and suggestion. You are right. This part existed serious weaknesses in discussion and lack of focus. After discussion, we rewrite and improve the "Discussion" part and also discuss our result with previous studies as: Lin 293-417:

The effects of Sphingobacterium sp. on C mineralization

Microorganisms are known to contribute to the mineralization and loss of SOC [START_REF] Schimel | Microbial control over carbon cycling in soil[END_REF].

Sphingobacterium sp. is a type of gram-negative bacteria. Likewise, previous studies [START_REF] Shankar | Saccharification of the pretreated 592 husks of corn, peanut and coffee cherry by the lignocellulolytic enzymes secreted by 127[END_REF][46] used Sphingobacterium sp. to decompose the agricultural byproducts and to biodegrade organic pollutants. The performance of Sphingobacterium sp. in both non-contaminated and Cd contaminated soils indicates its ability to mineralize carbon and tolerate metal stress. These abilities align with a similar study [47], who demonstrated that the inoculation of Sphingobacterium sp. can enhance the degradation of organic pollutant residues in Cd (0.30 mg•kg -1 ) contaminated soil. It also can be inferred from the aforementioned results that these functions of Sphingobacterium sp. were not dependent on the gut conditions of earthworms.

Additionally, the highest microbial biomass carbon content and the microbial quotient carbon in the CS treatment (Fig. 1C and Fig. 2E) were due to the inoculation of this strain, its adaption in the soil environment and substantial proliferation. However, this strong adaptive phenomenon was not observed in Cd-contaminated soil, where there is reduced microbial biomass carbon caused by metal stress. For instance, the adaptation process of Sphingobacterium sp. was observed in the early stage (60 days) in Cd-contaminated soil (0.3 mg•kg -1 ), with the strain population size gradually increasing after 90 days [47]. Nevertheless, this study only lasted for 20 days and more time is needed for Sphingobacterium sp. to complete the adaption processes under metal stress.

Earthworm survival, growth and species-specific effects on C and N transformation

Earthworm abundance and biomass decreased after 20 days of cultivation in both soils, especially in Cd-contaminated soil, consistent with findings from a previous study [29]. This decline may be attributed to soil nutrient limitation and the toxic effects of high Cd concentration on earthworms [48]. Regarding earthworm species, A. gracilis showed higher resistance to environmental stresses than E. fetida, probably owing to the different strategies employed by earthworms in metal handling and resistance. In a previous study, a toxicokinetic model was employed, which found that wild earthworm species were more specialized than E. fetida in balancing the trade-off between metal uptake and excretion processes based on their surrounding environment [49].

There are different effects of earthworms on soil C and N transformation in non-contaminated and Cd-contaminated soil. In non-contaminated soil with a high organic carbon content, earthworms alone facilitated soil organic carbon and nitrogen mineralization. This is consistent with the trend of carbon and nitrogen changes observed in vermicomposting [50],

presumably through the decomposition of organic matter by specific microorganisms under the unique conditions within the earthworm gut [51]. In addition, it can be found that earthworm activities increased the soil microbial quotient carbon compared to the control, indicating improved efficiency of soil organic matter utilization by microorganisms [52]. However, soil carbon stabilization in Cd-contaminated soil was probably due to earthworms consuming more organic matter to resist metal toxicity [START_REF] Ge | Eisenia fetida impact on cadmium 620 availability and distribution in specific components of the earthworm drilosphere[END_REF]. Moreover, it has been proved that gut peristalsis associates partly degraded organic matter with minerals [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF] and leads to formation of organic matter-rich aggregates [START_REF] Hong | How do 627 earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Guhra | Earthworm mucus contributes to the formation 630 of organo-mineral associations in soil[END_REF], which are conducive to SOC sequestration [7,[START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Nannoni | Soil properties and metal accumulation by earthworms in the 637 Siena urban area (Italy)[END_REF][START_REF] Nyenda | Changes in physicochemical properties on a 672 chronosequence of gold mine tailings[END_REF][60]. It was concluded that earthworms can slow down SOC mineralization in the presence of more minerals [52]. Therefore, earthworm activities reduced carbon mineralization in mineral-rich Cd-contaminated soil. Furthermore, this effect may also be explained by the fact that contaminated soils contain stable carbon that is not easily mineralized. The positive correlation between β-glucosidase and N-acetyl-β-D-glucosaminidase activity, demonstrated with PCA, also supports the notion that soil microorganisms tend to use the active carbon pool generated by the decomposition of humus formed by litter (Fig. 5A; Fig. S1), and this favors the accumulation of recalcitrant soil carbon pools.

The earthworm species have great impact on biogeochemical cycling through their feeding habits and cast properties [31]. In non-contaminated and Cd-contaminated soil, both E. fetida and A. gracilis perform silimar ecological functions; however, their performance is also influenced by earthworm survival strategies. In environments without metal stress, both species can enhance C and N mineralization. It is worth noting that E. fetida has a more pronounced effect on soil carbon and nitrogen mineralization than A. gracilis does (Fig. 1B andE; Fig. 2A andC).

This could be attributed to the preference of E. fetida for ingesting high-quality organic matter.

Their castings have a high level of particulate organic matter content and porosity that provides plentiful food and spaces for soil microorganisms [31], thereby accelerating the decomposition of organic matter [61][62][START_REF] Yadav | Role of earthworms in soil health and variablesinfluencing their 652 population dynamic: a review[END_REF]. On the contrary, the wild earthworm A. gracilis not only fed on soil organic matter, but also on more mineral particles. In addition, E. fetida found it easier to obtain food in non-contaminated soil with a relatively high organic matter content (Table 1). A high carbon to nitrogen ratio was favored for earthworm growth [START_REF] Fusilero | Weed management 654 systems and other factors affecting the earthworm population in a banana plantation[END_REF], so this species performed better.

In contrast, in Cd-contaminated soil, the mineralization processes of soil organic matter slow down. The wild earthworm species A. gracilis demonstrated better survival and had more significant effects on C stabilization than E. fetida (Table 2, Fig. 1 and Fig. 2). The low organic matter content in Cd-contaminated soils limited the decomposition functions of E. fetida.

Furthermore, earthworms need to consume more food to resistant Cd stress. Both species increases their food consumption to feed on more organic matter. In this process, earthworms were compelled to ingest more mineral particles in soils characterized by low organic matter (Table 1) and high soil mineral content. The gut-grazing process facilitated the thorough mixing and binding of organic matter with soil minerals [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF]. As for enzymes, the enzymes activity was influenced by earthworm species and metal stress. Likewise, in the previous study, earthworm activities suppressed β-glucosidase activity during the stimulation of C mineralization processes [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF]. It was speculated that when soil microorganisms could easily access the readily mineralized C released by earthworms, they would use a smaller portion of the carbon from enzyme-catalyzed decomposition of soil organic carbon. Enzyme activity only represents the concomitant consequence of earthworm activities but does not serve as the major driver of organic matter decomposition. It is also controlled by multiple factors such as food type, pH, temperature, gut transit time, metal stress, substrates, et al., [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF][START_REF] Karasov | Ecological physiology of diet and digestive 661 systems[END_REF][START_REF] Utobo | Soil enzymes as bioindicators of soil ecosystem status[END_REF].

The combined effects of earthworm and Sphingobacterium sp. on the balance between C and N stabilization and mineralization

The combination processes showed that Sphingobacterium sp. assisted earthworms in further promoting carbon and nitrogen mineralization in non-contaminated soils (Fig. 4A andC) and carbon stabilization in Cd-contaminated soils (Fig. 5A andC), as reflected by the increased contents of mineralization (DOC, AN) and the stabilization indicators (SOC), respectively. It was also found that inoculated earthworm can process organically polluted water and excess sludge, and Sphingobacterium, known for its high ability to degrade organic matter, is one of the most abundant microbes in earthworm castings [START_REF] Liu | Effect of earthworms on the performance 666 and microbial communities of excess sludge treatment process in vermifilter[END_REF]. Nevertheless, our study demonstrated that external addition of Sphingobacterium sp. can help earthworms adapt more easily to environment and exert their ecological functions. Despite having similar effects when used in combination or when being inoculated alone, the strategies of the two species differed in non-contaminated and Cd-contaminated soil (Fig. 4D). Compared with treatments that inoculate earthworms alone, CSA and CSE treatments further activated INV and PRO activities in non-contaminated soil (Fig. 3 and Fig. 4D); On the other hand, SSA further activated INV activities and inhibited PRO activities, while SSE further inhibited URE activities (Fig. 3 and Fig. 5D). It is well known that soil enzyme activity is mainly derived from microorganisms and can reflect microbial activity [START_REF] Li | Microbial biomass, 669 enzyme and mineralization activity in relation to soil organic C, N and P turnover influenced by 670 acid metal stress[END_REF]. Inoculation of Sphingobacterium sp. may stimulate microbial activities in the soil and the earthworm gut in non-contaminated soil [16]. In Cd-contaminated soil, it may represent the natural response of earthworms to the inoculation of Sphingobacterium sp. and metal stress. Compared to non-contaminated soil, metal stress induced inhibition of PRO activities in the treatment with A. gracilis inoculation and URE activities in the treatment with E. fetida inoculation, indirectly demonstrating distinct coping strategies for metal stress among earthworm species. Given that researchers have [START_REF] Akinnuoye-Adelabu | Effect of 673 redworm extracts against Fusarium root rot during wheat seedling emergence[END_REF] found that the vermiwash and mucus extracted from earthworms can greatly inhibit the growth of pathogenic fungi, it is also possible that the mucus compounds changed due to metal pollutant stress in this study, such as secretion of specific organic substances to protect itself from physiological damage, while inhibiting the microbial activities. The underlying mechanisms are still not clear and require further research.

In these processes, earthworm activities dominated the soil carbon and nitrogen mineralization processes, while Sphingobacterium sp. played a supporting role in combined processes. When the effects of earthworm and Sphingobacterium sp. on carbon and nitrogen were consistent, they enhanced carbon mineralization; otherwise they undermined this process. Hence, in non-contaminated soil, carbon mineralization was observed. While in Cd-contaminated soil, carbon storage was more prevalent due to the stronger effect of earthworms on stabilization than Sphingobacterium sp. on mineralization. This is further confirmed by PCA which demonstrated that earthworms play a critical role in soil organic carbon decomposition processes, following the same trend as inoculation with earthworm alone and in combination with Sphingobacterium sp.

(Fig. 4A andC; Fig. 5A andC). Additionally, in this system, a strong interaction existed between earthworms and Sphingobacterium sp.. Earthworm activities under the combined process dramatically decreased microbial biomass carbon compared to Sphingobacterium sp. activities alone. It is suggested that earthworms contribute to maintaining the ecosystem balance and stabilizing the microbial community structure through feeding [START_REF] Zhang | Influence of the earthworm Pheretima guillelmi on soil 676 microbial biomass and activity[END_REF] and regulating the allocation of active organic carbon and dissolved organic carbon pools [START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Yu | Effects of straw application and earthworm inoculation on 679 soil labile organic carbon[END_REF]. Therefore, the rapid proliferation of Sphingobacterium sp. was prevented in CSE and CAS treatments. It was further confirmed by the significantly lower microbial quotient carbon in treatment CSE and CSA compared to treatment CS (Fig. 2E). It can be deduced that earthworms may reduce the conversion efficiency of soil microorganisms in utilizing carbon sources into microbial biomass carbon [52] or they can intensely feed and graze on microbes [10,[START_REF] Vafa | Sewage sludge application strongly modifies earthworm 681 impact on microbial and biochemical attributes in a semi-arid calcareous soil from Iran[END_REF][START_REF] Edwards | Interactions between earthworms, microorganisms, and 684 other invertebrates[END_REF][START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF]. Additionally, the different impact of earthworm species may be caused by E. fetida being more conducive to the survival and growth of G -bacterial communities [START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF], thus promoting Sphingobacterium sp. to assist earthworms in carbon mineralization. Based on the above, another underlying factor was postulated that Sphingobacterium sp. may assist earthworms in mineralizing soil organic carbon and nitrogen by stimulating the microbial activities in the earthworm gut. The subsequent studies need to focus on the earthworm gut microbial to reveal the internal mechanisms.

Writing and grammar.

There are a number of sentences that need improvement. For example, 1) The clean soil in this study means non-Cd-contaminated soil. Therefore, it should be better to use the Cd-contaminated and non-contaminated soils to describe these two soils.

Answer: Thanks for your comment and suggestion. We agree with you and have been replaced "clean soil and contaminated soil" to "non-contaminated soil and Cd-contaminated soil" to describe these two soils.

2) INV, inverse activity (in the figure caption of Fig. 3) has a spelling error.

Answer: Thanks for your comment. This word in the full text has been revised.

3) The name of earthworm in this study should be "E. fetida" and "A. gracilis", not "E.fetida" and "A.gracilis".

Answer: Thanks for your comment. Sorry about this mistake. The abbreviation format of earthworm species in the full-text has been revised as "E. fetida" and "A. gracilis".

4) There should be a space before the parentheses and brackets. Many similar typing mistakes can be found through the manuscript. Please carefully check and revise them.

Answer: Thanks for your reminding and suggestion. The full-text has been checked and revised.

6.

Please find a professional editing service or a native speaker of English to check the language.

Answer: Thanks for your suggestion. This manuscript of new version has been checked and polished by the professional institute. 2) Table 2. The significance difference in biomass on the 20th day (a, b) was mislabeled.

Answer: Thanks for your suggestion. We revised the data format and check the significance between the treatments, like below: 8. Line 81-82. "Earthworm selective feeding activities have a significant impact on soil carbon and nitrogen cycling in gut [6]." What do you mean "soil carbon and nitrogen cycling in gut"? Please clarify.

Answer: Thanks for your comment. Sorry for our omission. It should be earthworm selective feeding activities have a significant impact on soil carbon and nitrogen cycling. We have revised as:

Line 89-90: Earthworm's preferential feeding and concentration processes have a substantial impact soil carbon and nitrogen cycling [7]. Importantly, it is worth to be noticed that the different effects of them and trying to convert laboratory tests into field experiments. We clarified in the "Introduction" part as:

Line 112-117: In addition, different earthworm species and ecological categories have varying effects on soil organic matter degradation and soil C and N cycles [31]. The species of the experimental model earthworm E. fetida and local wild epigeic species may have different survival strategies that affect microbial biomass or activities, resulting in their different performances in soil carbon and nitrogen cycles in non-contaminated and Cd-contaminated soil.

10. Line 139. The abbreviation of Amynthas gracilis is in a wrong format. A space should be added between the genus name and the species name. Many similar typing mistakes can be found through the manuscript. Please carefully check and revise them.

Answer: Thanks for your comment. The abbreviation format of earthworm species has been revised as "E. fetida" and "A. gracilis". In addition, the full-text has been checked and revised.

11. Line 144-146. What is microbial CM0827 Propionibacterium sp. medium? Please clarify why use this kind of medium. In addition, there should be no commas followed by consist of.

Answer: Thanks for your comment and suggestion. The CM0827 Propionibacterium sp.

medium means medium with the number CM0827 and the name Propionibacterium sp. medium, which can support the growth of gram-negative bacteria. Thanks for your kind reminding. We have deleted the punctuation followed by consist of.

12. Line 147. The pH and OD405 value 1.45 should be placed after the recipe of medium and before the incubation of bacteria in the tested soils, respectively. By the way, what do you mean "set aside"?

Please clarify.

Answer: Thanks for your comment and suggestion. Sorry to describe not clear. We already modified it as:

Line 165-168: CM0827 Propionibacterium sp. medium, which consisted of tryptone 15 g, L-cystine 0.5 g, yeast extract 5 g, sodium thioglycolate 0.5 g, glucose 5 g, agar 0.7 g, sodium chloride 2.5 g, resazurin 0.001 g, distilled water 1 L, was employed to cultural Sphingobacterium sp. and was set at pH 7.0.

In addition, the means of "set aside" is "prepared the experiment materials waiting to be used in the subsequent experiments". We already revised it as:

Line 168-169: The strain was incubated at a constant temperature of 30°C for 2 days before inoculation into the soil.

13. Line 180-182. This sentence needs revision. For example, "Total cadmium (Cd) content" of whom? What was the certified standard material? Please provide.

Answer: Thanks for your comment. We added the explanation of DTPA, the determination methods of DTPA and total Cd content of soil, and the certified standard materials. This part be revised as:

Line 203-213: 2.4 Total and DTPA extractable Cadmium content Total cadmium (Cd) in the soils was extracted by microwave digestion with aqua regia (HNO3:HCl (v/v) = 1:3). The diethylenetriaminepentaacetic acid (DTPA) extractable form was regarded as bioavailable metals and toxic. DTPA-extractable Cd was extracted with DTPA extractant at a Soil:DTPA extractant ratio of 5:25 (g/mL), which included a solution of 5 Mm DTPA and 10 mM CaCl2. The extracted suspensions were analyzed for total cadmium (Cd) and DTPA-extractable contents using inductively coupled plasma-mass spectrometry (ICP-MS, 7700ce, Agilent Technologies, Santa Clara, CA, USA) [43]. GBW07455 (GSS-26) was employed as certified reference material for soil analyses, and the measured concentrations were within 98% of the reported certified concentrations for Cd. Quality control measures included three repetitions for each soil and three blanks for each batch to ensure the quality of the analysis.

14.

Line 205-207. This sentence is very confusing. Please rewrite it.

Answer: Thanks for your comment. We revised the data format order to show the contents of Line 205-207 in Table 2, and deleted Line 205-207. It will be clearer and easier to understand than before.

15. Line 251-253. Technically, INV activity is one of the enzymes activity. Please revise this sentence.

Answer: Thanks for your comment. This sentence really induced the misunderstanding. We deleted ", but greater variation in the effect on INV activities." in Line 252-253 after reconsidering.

16.

Line 394-399. This sentence is too long and hard to digest. Please rewrite.

Answer: Thanks for your comment. Because we reorganized the discussion part, the relevant contents were revised as:

Line 327-328: However, soil carbon stabilization in Cd-contaminated soil was probably due to earthworms consuming more organic matter to resist metal toxicity [START_REF] Ge | Eisenia fetida impact on cadmium 620 availability and distribution in specific components of the earthworm drilosphere[END_REF]. Moreover, it has been proved that gut peristalsis associates partly degraded organic matter with minerals [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF] and leads to formation of organic matter-rich aggregates [START_REF] Hong | How do 627 earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Guhra | Earthworm mucus contributes to the formation 630 of organo-mineral associations in soil[END_REF], which are conducive to SOC sequestration [7,[START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Nannoni | Soil properties and metal accumulation by earthworms in the 637 Siena urban area (Italy)[END_REF][START_REF] Nyenda | Changes in physicochemical properties on a 672 chronosequence of gold mine tailings[END_REF][60]. It was concluded that earthworms can slow down SOC mineralization in the presence of more minerals [52]. Therefore, earthworm activities reduced carbon mineralization in mineral-rich Cd-contaminated soil.

#AU_EJSOBI#

To ensure this email reaches the intended recipient, please do not delete the above code carbon and nitrogen cycles. In this study, earthworms (Eisenia fetida, Amynthas gracilis) and Sphingobacterium sp. were inoculated in clean and contaminated soils under laboratory condition for 20 days. We investigated their single or combined effects on carbon and nitrogen forms (organic carbon, total Nitrogen, dissolved organic carbon (DOC), alkali-hydrolyzable nitrogen (AN), significantly influence soil carbon and nitrogen contents [13]. These processes is the 80 result of complex interactions between soil organisms. Earthworm selective feeding 81 activities have a significant impact on soil carbon and nitrogen cycling in gut [6].

microbial
82 Earthworm bioturbation (burrowing, feeding and excretion (casts and surface mucus))

83 affect directly or indirectly soil microbial communities and soil enzyme activities [7][8][9]. carbon and nitrogen metabolism in soils [29]. Fluorescein diacetate hydrolysis (FDA)

112 is an enzyme that can reflect the total microbial activity of soil [30]. Therefore, the and total nitrogen (TN) was analyzed by the pipette [31], the dichromate oxidation [32],

176 alkali N-proliferation [33] and Kjeldahl [32] methods, respectively;Microbial biomass extraction method [34,35]; Dissolved organic carbon (DOC) content was extracted with 179 deionized water (1:5) and determined [15]. Total cadmium (Cd) content was prepared 180 and determined by microwave digestion and ICP-MS [36], and the measured 181 concentrations of referred samples within 98% of certified concentrations. Three 182 repetitions for each soil were also implemented to ensure the quality of analysis. activities was determined by sodium phenolate colorimetric assay, titration and 186 colourimetric using fluorescein methods [37], respectively.

The content of carbon and nitrogen fractions 208

The soil carbon fraction contents were shown in Fig. 1a-c

224

The content of nitrogen fractions can be found in Fig. 1d-f Soil carbon and nitrogen fraction ratios are shown in Fig. 2. In both soils, the 234 ratios showed consistency with the changes in the corresponding carbon and nitrogen 235 fractions.

236

The ratio of DOC:SOC followed the same trend as DOC, increasing to 2.93% 237 and 1.94% in the CSE and CSA treatments, respectively (Fig. 2a). The ratio of C:N 238 significantly decreased by 23.6%, 23.6%, 24.0%, 22.6% and 14.4% in the CS, CE, CA, There was no significant change in FDA activities (p>0.05).

279

In contaminated soils, two principal components (PCs) were identified in the 280 PCA, representing 43.7% of the total variance (Fig. 4c-d). PC1 and PC2 explained 26.6% 281 and 17.1% of the total variance, respectively. There were significant differences 282 between the treatments, indicating that earthworms and Sphingobacterium sp. of high Cd concentration on earthworms [39]. In terms of earthworm species, A.gracilis 297 showed higher resistant to environmental stresses than E.fetida, probably owing to the adaptability of wild earthworm species in soil with limited organic matters. In clean soils with high organic carbon, both earthworm and Sphingobacterium 302 sp. alone significantly facilitated the mineralization of organic carbon (Fig. 1b, Fig. 2b)

303
and is consistent with the trend of carbon and nitrogen changes in vermicomposting by 304 [START_REF] Vance | An extraction method for measuring soil microbial 577 biomass C[END_REF]. Both species of earthworms significantly increased nitrogen mineralization (p< 305 0.05) (Fig. 1e), presumably through digestion in the earthworm gut, which promotes 306 the mineralization of organic nitrogen. A study investigated the changes in the gut 307 microbiota and functional characteristics of earthworms with the chronological 308 sequence of cultivated soils, revealing that tillage disturbance increases the formation 309 of functional genes that promote nutrient cycling in the earthworm gut microbiota [START_REF] Dai | Influence of heavy metals on C and N 580 mineralisation and microbial biomass in Zn-, Pb-, Cu-, and Cd-contaminated soils[END_REF].

310

The combination of the two species of earthworm with Sphingobacterium sp. also and Sphingobacterium sp. activities had non-significant effect, presumably due to 348 nutrient and metal pollution stress [48], which in turn reduces its ecological role. The soil enzyme activity in both clean and Cd-contaminated soils showed in Fig. results of most studies [49,50]. However, it did not affect the soil carbon and nitrogen 357 mineralization. Similarly, a study found that acid and alkaline phosphatase activities 358 were decreased in earthworm casts but inorganic phosphorus in the soil was increased 359 [8]. This also suggests that enzyme activity is affected by a variety of factors such as 360 food type, pH, temperature, gut transit time, etc., [51]. Besides, enzymes only play a 361 catalytic role and do not completely determine soil matter mineralization, which needs 362 to be analyzed in the context of multiple biological and environmental factors.

363

In contaminated soil, earthworms and Sphingobacterium sp. activities decreased the activities of invertase, N-acetyl-β-D glucosaminidase and urease, but there was no 365 obvious regularity among treatments (Fig. 3b-d). It is possibly due to that soil exhibited 366 carbon storage and the corresponding mineralizing enzyme activities were reduced 367 under the activities of earthworms. Or these enzyme activities were intimately related 368 to soil water-soluble organic carbon and microbials, i.e., metal stress and low soil 369 fertility, and soil fauna activities in turn reduced the soil microbiota [52,[START_REF] Ge | Eisenia fetida impact on cadmium 620 availability and distribution in specific components of the earthworm drilosphere[END_REF]. In addition, 370 metal can inhibite soil urease and fluorescein diacetate hydrolytic activity, and 371 postulated that the mechanism may be that Cd 2+ exchanges with Mg 2+ , resulting in 372 blockage of protein synthesis, which in turn inhibits the growth and proliferation and 373 decreases enzyme synthesis and secretion in microorganisms, and ultimately leads to 374 decreased soil enzyme activities [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF].

375

In terms of earthworm species, soil enzyme activities in clean soil with E.fetida 376 participating were generally higher than those of A.gracilis. In contaminated soils,

377

A.gracilis increased N-acetyl-β-D glucosaminidase, protease, and urease activities 378 individual and combined, which may be related to that A.gracilis is wild species with 379 strong resistance to environment stress. Moreover, A.gracilis influenced fluorescein 380 diacetate hydrolytic to a greater extent, both individually and combinatorially in two 381 soils, presumably as a result of differences in feeding habits of different species 382 [START_REF] Hong | How do 627 earthworms influence organic matter quantity and quality in tropical soils?[END_REF]10,[START_REF] Guhra | Earthworm mucus contributes to the formation 630 of organo-mineral associations in soil[END_REF]. Earthworms preferred to feed on microorganisms as secondary food source 383 in soils where organic matter is difficult to degrade [8]. The body fluids of earthworms 384 may contain bacteriostatic substances [START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Nannoni | Soil properties and metal accumulation by earthworms in the 637 Siena urban area (Italy)[END_REF], resulting in a lower bacterial content in 385 their gut and excreta than in original soil. matter that is stable and not easily mineralized by organisms [START_REF] Nyenda | Changes in physicochemical properties on a 672 chronosequence of gold mine tailings[END_REF]. Among earthworm 403 species, E.fetida had a stronger effect on soil carbon and nitrogen mineralization than A.gracilis (Fig. 1b), which may be related that E.fetida favors ingestion of easily 405 available organic matter, accelerating the mineralization and decomposition of organic 406 matter [60,61]. By contrast, A.gracilis mainly fed on soil mineral particles and organic 407 matter, facilitating the mixing and bind of organic matter with soil minerals. In addition, the soil of this study (Table 1), E. fetida was easier to obtain food in clean soil with 409 relatively high organic matter content and the high carbon to nitrogen ratio was favored 410 for earthworm growth [62], so this species performed better. Unfortunately, their 411 ecological effects were limited by difficulties in access to food in Cd-contaminated soils 412 with low organic matter content(Table 1). Additionally, only parts of treatment 413 increased soil enzyme activities. Mostly treatment enzymes had no significant different 414 compared with control treatment and showed weak inhibited by earthworm activities.

415

This phenomenon also can be founded in PCA analysis (Fig. 4).  Earthworm activities dominated the C and N turnover in this strain-earthworm-soil system. 32

Introduction 75

Soil serves as both an important source and sink for terrestrial carbon and nitrogen in the 76 biosphere. Various soil organisms play essential roles in soil carbon and nitrogen cycles [1,2]. The 77 interactive processes involving soil organisms and restoration of soil carbon and nitrogen stocks in 78 sustainable soil management have garnered widespread attention [3,4]. However, soil metal pollution 79 is increasingly emerging as a serious environmental and social concern, potentially resulting in 80 changes in the interactions among soil organisms and affecting soil carbon and nitrogen cycles [5]. It 81 is of great ecological importance to understand the interactive relationships among soil organisms in 82 the context of the carbon and nitrogen cycle, particularly under non-contaminated and metal-83 contamination stress. 84

As "soil ecosystem engineers", earthworm can degrade soil organic matter and significantly 85 influence the forms and contents of soil carbon and nitrogen [6]. Earthworm's preferential feeding and 86 concentration processes have a substantial impact soil carbon and nitrogen cycling [7]. Their 87 bioturbation activities, including burrowing, feeding, and excretion (casts and surface mucus), affects 88 the soil carbon and nitrogen distribution within soil profile [8][9][10][11]. These effects result from complex 89 mutualism interactions between earthworms and microorganisms [7,[12][13]. Previous studies have 90 established connections between earthworms and rhizosphere growth-promoting bacteria or specific 91 arbuscular mycorrhizal fungi to enhance nutrient availability or nitrogen storage in soil [14][15]. 92

Moreover, the collaboration of indigenous earthworms and gut bacteria has been employed to 93 remediate metal contaminated soil and has shown superiority compared to chemical amendments [16]. 94

Likewise, combining earthworms with dominate gut bacteria taxa to enhance organic matter 95 decomposition is also advantageous, because it is more readily accepted by earthworms. In our 96 previous studies, Sphingobacterium sp. was identified as a dominant bacterial taxon in the earthworm 97 gut [17]. Sphingobacterium sp., a gram-negative bacterium, exhibits a wide range of nutrient sources, 98 high adaptability, and a broad ecological distribution [18][19][20]. In practice, it has been used to 99 decompose agricultural waste and degrade organic pollutants (e.g. alkanes and polycyclic aromatic 100 hydrocarbons) in soil [START_REF] Dong | Manipulation of microbial community in the rhizosphere alleviates the 536 replanting issues in Panax ginseng[END_REF][22][23][24][25][26]. Therefore, this decomposition ability of earthworms probably derives 101 from symbiotic microorganisms in their guts. Considering that earthworms and Sphingobacterium sp. 102 impact organic matter decomposition and the latter was dominant bacterial taxon in earthworm gut, a 103 synergistic relationship may exist, enhancing soil organic matter decomposition. 104 However, under environment stress, such as metal contamination, the activities and complex 105 interactions of them may be suppressed. Specifically, metal contamination can induce biomass loss, 106 reduce survival rate, cause irreversible physiological damage, decrease the stability of gut bacterial 107 network, and dampen its ecological functions [27][28][29]. Similarly, the inhibition effects also impact soil 108 microorganisms, leading to reduced soil enzyme functions critical for organic matter cycling [30]. In 109 addition, different earthworm species and ecological categories have varying effects on soil organic 110 matter degradation and soil C and N cycles [31]. The species of the experimental model earthworm E. 111 fetida and local wild epigeic species may have different survival strategies that affect microbial 112 biomass or activities, resulting in their different performances in soil carbon and nitrogen cycles in 113 non-contaminated and Cd-contaminated soil. 114 Soil C and N cycles involve a series of biological and biochemical processes. Soil carbon and 115 nitrogen forms, dissolved organic carbon (DOC), alkali-hydrolysable nitrogen (AN), microbial 116 biomass carbon and nitrogen (MBC and MBN) have been considered as the key indicators for 117 assessing the status of the soil carbon and nitrogen cycles. The ratios of these forms can further provide 118 a more intuitive representation of the overall mineralization or sequestration of carbon and nitrogen. 119

Besides, soil enzymes are metabolic drivers for soil organisms, and their activity can, to some extent, 120 reflect the changes in the soil microbial community [32], and the intensity of soil organic matter 121 decomposition and nutrient transformation [33]. Studies on the influence of enzymes on carbon and 122 nitrogen transformation have mainly focused on β-glucosidases (BG) and invertase (INV), which are 123 involved in the decomposition of labile cellulose, sucrose, and carbohydrate polymers. N-acetyl-124 glucosaminidase (NAG), urease (URE), and proteases (PRO) are involved in the hydrolysis of 125 chitooligosaccharide, urea and protein. These enzymes mainly participate in terminal catalytic 126 reactions or intra-systemic enzymatic reactions, which can reflect the level of carbon and nitrogen 127 metabolism in soils [34]. Furthermore, fluorescein diacetate hydrolysis (FDA) is an enzyme that can 128 indicate the total microbial activity in the soil [35]. Therefore, the effects of earthworm inoculation on 129

Materials and methods

uptake and excretion processes based on their surrounding environment [49]. 316

There are different effects of earthworms on soil C and N transformation in non-contaminated 317 and Cd-contaminated soil. In non-contaminated soil with a high organic carbon content, earthworms 318 alone facilitated soil organic carbon and nitrogen mineralization. This is consistent with the trend of 319 carbon and nitrogen changes observed in vermicomposting [50], presumably through the 320 decomposition of organic matter by specific microorganisms under the unique conditions within the 321 earthworm gut [51]. In addition, it can be found that earthworm activities increased the soil microbial 322 quotient carbon compared to the control, indicating improved efficiency of soil organic matter 323 utilization by microorganisms [52]. However, soil carbon stabilization in Cd-contaminated soil was 324 probably due to earthworms consuming more organic matter to resist metal toxicity [START_REF] Ge | Eisenia fetida impact on cadmium 620 availability and distribution in specific components of the earthworm drilosphere[END_REF]. Moreover, it 325 has been proved that gut peristalsis associates partly degraded organic matter with minerals [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF] and 326 leads to formation of organic matter-rich aggregates [START_REF] Hong | How do 627 earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Guhra | Earthworm mucus contributes to the formation 630 of organo-mineral associations in soil[END_REF], which are conducive to SOC 327 sequestration [7,[START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Nannoni | Soil properties and metal accumulation by earthworms in the 637 Siena urban area (Italy)[END_REF][START_REF] Nyenda | Changes in physicochemical properties on a 672 chronosequence of gold mine tailings[END_REF][60]. It was concluded that earthworms can slow down SOC mineralization in the 328 presence of more minerals [52]. Therefore, earthworm activities reduced carbon mineralization in 329 mineral-rich Cd-contaminated soil. Furthermore, this effect may also be explained by the fact that 330 contaminated soils contain stable carbon that is not easily mineralized. The positive correlation 331 between β-glucosidase and N-acetyl-β-D-glucosaminidase activity, demonstrated with PCA, also 332 supports the notion that soil microorganisms tend to use the active carbon pool generated by the 333 decomposition of humus formed by litter (Fig. 5A; Fig. S1), and this favors the accumulation of 334 recalcitrant soil carbon pools. 335

The earthworm species have great impact on biogeochemical cycling through their feeding 336 habits and cast properties [31]. In non-contaminated and Cd-contaminated soil, both E. fetida and A. 337 gracilis perform silimar ecological functions; however, their performance is also influenced by 338 earthworm survival strategies. In environments without metal stress, both species can enhance C and 339 N mineralization. It is worth noting that E. fetida has a more pronounced effect on soil carbon and 340 nitrogen mineralization than A. gracilis does (Fig. 1B andE; Fig. 2A andC). This could be attributed 341 to the preference of E. fetida for ingesting high-quality organic matter. Their castings have a high level 342 of particulate organic matter content and porosity that provides plentiful food and spaces for soil 343 microorganisms [31], thereby accelerating the decomposition of organic matter [61][62][START_REF] Yadav | Role of earthworms in soil health and variablesinfluencing their 652 population dynamic: a review[END_REF]. On the 344 contrary, the wild earthworm A. gracilis not only fed on soil organic matter, but also on more mineral 345 particles. In addition, E. fetida found it easier to obtain food in non-contaminated soil with a relatively 346 high organic matter content (Table 1). A high carbon to nitrogen ratio was favored for earthworm processes of soil organic matter slow down. The wild earthworm species A. gracilis demonstrated 349 better survival and had more significant effects on C stabilization than E. fetida (Table 2, Fig. 1 and 350 Fig. 2). The low organic matter content in Cd-contaminated soils limited the decomposition functions 351 of E. fetida. Furthermore, earthworms need to consume more food to resistant Cd stress. Both species 352 increases their food consumption to feed on more organic matter. In this process, earthworms were 353 compelled to ingest more mineral particles in soils characterized by low organic matter (Table 1) and 354 high soil mineral content. The gut-grazing process facilitated the thorough mixing and binding of 355 organic matter with soil minerals [START_REF] Barthod | 623 How do earthworms affect organic matter decomposition in the presence of clay-sized 624 minerals?[END_REF]. As for enzymes, the enzymes activity was influenced by 356 earthworm species and metal stress. Likewise, in the previous study, earthworm activities suppressed 357 β-glucosidase activity during the stimulation of C mineralization processes [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF]. It was speculated that 358 when soil microorganisms could easily access the readily mineralized C released by earthworms, they 359 would use a smaller portion of the carbon from enzyme-catalyzed decomposition of soil organic carbon. 360

Enzyme activity only represents the concomitant consequence of earthworm activities but does not 361 serve as the major driver of organic matter decomposition. It is also controlled by multiple factors such 362 as food type, pH, temperature, gut transit time, metal stress, substrates, et al., [START_REF] Lv | Earthworm Pontoscolex 657 corethrurus stimulated soil CO2 emission by enhancing substrate availability rather than 658 changing microbiota community structure[END_REF][START_REF] Karasov | Ecological physiology of diet and digestive 661 systems[END_REF][START_REF] Utobo | Soil enzymes as bioindicators of soil ecosystem status[END_REF]. The combination processes showed that Sphingobacterium sp. assisted earthworms in further 366 promoting carbon and nitrogen mineralization in non-contaminated soils (Fig. 4A andC) and carbon 367 stabilization in Cd-contaminated soils (Fig. 5A andC), as reflected by the increased contents of 368 mineralization (DOC, AN) and the stabilization indicators (SOC), respectively. It was also found that 369 inoculated earthworm can process organically polluted water and excess sludge, and 370 Sphingobacterium, known for its high ability to degrade organic matter, is one of the most abundant 371 microbes in earthworm castings [START_REF] Liu | Effect of earthworms on the performance 666 and microbial communities of excess sludge treatment process in vermifilter[END_REF]. Nevertheless, our study demonstrated that external addition of 372 Sphingobacterium sp. can help earthworms adapt more easily to environment and exert their ecological 373 functions. Despite having similar effects when used in combination or when being inoculated alone, 374 the strategies of the two species differed in non-contaminated and Cd-contaminated soil (Fig. 4D). 375

Compared with treatments that inoculate earthworms alone, CSA and CSE treatments further activated 376

INV and PRO activities in non-contaminated soil (Fig. 3 and Fig. 4D); On the other hand, SSA further 377 activated INV activities and inhibited PRO activities, while SSE further inhibited URE activities (Fig. 378 3 and Fig. 5D). It is well known that soil enzyme activity is mainly derived from microorganisms and 379 can reflect microbial activity [START_REF] Li | Microbial biomass, 669 enzyme and mineralization activity in relation to soil organic C, N and P turnover influenced by 670 acid metal stress[END_REF]. Inoculation of Sphingobacterium sp. may stimulate microbial activities in the soil and the earthworm gut in non-contaminated soil [16]. In Cd-contaminated soil, it 381 may represent the natural response of earthworms to the inoculation of Sphingobacterium sp. and metal 382 stress. Compared to non-contaminated soil, metal stress induced inhibition of PRO activities in the 383 treatment with A. gracilis inoculation and URE activities in the treatment with E. fetida inoculation, 384 indirectly demonstrating distinct coping strategies for metal stress among earthworm species. Given 385 that researchers have [START_REF] Akinnuoye-Adelabu | Effect of 673 redworm extracts against Fusarium root rot during wheat seedling emergence[END_REF] found that the vermiwash and mucus extracted from earthworms can greatly 386 inhibit the growth of pathogenic fungi, it is also possible that the mucus compounds changed due to 387 metal pollutant stress in this study, such as secretion of specific organic substances to protect itself 388 from physiological damage, while inhibiting the microbial activities. The underlying mechanisms are 389 still not clear and require further research. 390

In these processes, earthworm activities dominated the soil carbon and nitrogen mineralization 391 processes, while Sphingobacterium sp. played a supporting role in combined processes. When the 392 effects of earthworm and Sphingobacterium sp. on carbon and nitrogen were consistent, they enhanced 393 carbon mineralization; otherwise they undermined this process. Hence, in non-contaminated soil, 394 carbon mineralization was observed. While in Cd-contaminated soil, carbon storage was more 395 prevalent due to the stronger effect of earthworms on stabilization than Sphingobacterium sp. on 396 mineralization. This is further confirmed by PCA which demonstrated that earthworms play a critical 397 role in soil organic carbon decomposition processes, following the same trend as inoculation with 398 earthworm alone and in combination with Sphingobacterium sp. (Fig. 4A andC; Fig. 5A andC). 399

Additionally, in this system, a strong interaction existed between earthworms and Sphingobacterium 400 sp.. Earthworm activities under the combined process dramatically decreased microbial biomass 401 carbon compared to Sphingobacterium sp. activities alone. It is suggested that earthworms contribute 402 to maintaining the ecosystem balance and stabilizing the microbial community structure through 403 feeding [START_REF] Zhang | Influence of the earthworm Pheretima guillelmi on soil 676 microbial biomass and activity[END_REF] and regulating the allocation of active organic carbon and dissolved organic carbon pools 404 [START_REF] Zhang | Earthworms facilitate carbon sequestration through unequal amplification of 634 carbon stabilization compared with mineralization[END_REF][START_REF] Yu | Effects of straw application and earthworm inoculation on 679 soil labile organic carbon[END_REF]. Therefore, the rapid proliferation of Sphingobacterium sp. was prevented in CSE and CAS 405 treatments. It was further confirmed by the significantly lower microbial quotient carbon in treatment 406 CSE and CSA compared to treatment CS (Fig. 2E). It can be deduced that earthworms may reduce the 407 conversion efficiency of soil microorganisms in utilizing carbon sources into microbial biomass carbon 408 [52] or they can intensely feed and graze on microbes [10,[START_REF] Vafa | Sewage sludge application strongly modifies earthworm 681 impact on microbial and biochemical attributes in a semi-arid calcareous soil from Iran[END_REF][START_REF] Edwards | Interactions between earthworms, microorganisms, and 684 other invertebrates[END_REF][START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF]. Additionally, the different impact 409 of earthworm species may be caused by E. fetida being more conducive to the survival and growth of 410 G -bacterial communities [START_REF] Tibihenda | Ecologically different earthworm species are the driving force of 688 microbial hotspots influencing Pb uptake by the leafy vegetable Brassica campestris[END_REF], thus promoting Sphingobacterium sp. to assist earthworms in carbon 411 mineralization. Based on the above, another underlying factor was postulated that Sphingobacterium 412 sp. may assist earthworms in mineralizing soil organic carbon and nitrogen by stimulating the microbial activities in the earthworm gut. The subsequent studies need to focus on the earthworm gut 414 microbial to reveal the internal mechanisms. 415

Conclusion

  and combined effects of earthworms and Sphingobacterium sp. on soil organic C, N forms and enzyme activities in non-contaminated and Cd-contaminated soil Reviewer #1:

  approximately 8 individual E. fetida (~ 0.37 g•ind -1 ) or 5 individual A. gracilis (~ 0.60 g•ind -1 ).". In previous study, the average weight of E. fetida is around 0.4 g•ind -1 (Yue et al., 2019). Therefore, we think it is in normal range of biomass of E. fetida. Reference: S.Z. Yue, H.Q. Zhang, H.Y. Zhen, Z.Q. Lin, Y.H. Qiao, Selenium accumulation, speciation and bioaccessibility in selenium-enriched earthworm (Eisenia fetida), Microchemical Journal 145 (2019) 1-8, https://doi.org/10.1016/j.microc.2018.10.015. 4. Line 173, What is DTPA Cd, and how to measure it?

  figure label as:

7 .Fig. 1 .Fig. 2 .

 712 abbreviate the titles of vertical coordinates.Answer: Thanks for your comment and suggestion. The text of all the figures was enlarged.We replaced the abbreviation by the full name in the titles of vertical coordinates. In fig.2, there do not have more space to put the full name of the ratio such as DOC:DON, C:N, AN:TN, and MBC:MBN. Therefore, we keep same vertical coordinates with before and noted it under the figure. Thanks for your understanding. Like below:

Fig. 3 .

 3 Fig. 3. Characterization of soil enzyme activity. Results are expressed as the mean value ± SE (n = 3). Different lowercase letters indicate significantly differences (P < 0.05) among the treatments.

9 .

 9 Line 138-139. Why did the authors select two epigeic species of earthworms in this study? Please clarify. Answer: Thanks for your comment. The different species and ecological categories have different impacts on soil organic matter decomposition and soil C and N cycles. The species of the experimental model earthworm E. fetida and local wild epigeic species may have different survival strategies that affect microbial biomass or activities, resulting in their different performances in soil carbon and nitrogen cycles in non-contaminated and Cd-contaminated soil.

33 44 (

 44 Earthworm and Sphingobacterium sp. are both characterized by their strong 36 decomposition ability of organic compound and broad distribution in soil. Little is 37 known about their interaction on soil organic matter decomposition and whether 38 Sphingobacterium sp. could assist earthworms with carbon and nitrogen cycles. In this 39 study, earthworms (Eisenia fetida, Amynthas gracilis) and Sphingobacterium sp. were 40 inoculated in clean and contaminated soils under laboratory condition for 20 days. We 41 investigated their single or combined effects on carbon and nitrogen forms (organic 42 carbon, total Nitrogen, dissolved organic carbon (DOC), alkali-hydrolyzable nitrogen 43 (AN), microbial biomass carbon and nitrogen) and C-and N-related enzyme activities ), protease(PRO) and fluorescein diacetate hydrolysis (FDA)), 46 evaluating their interaction on soil C and N cycles.

53 fetida+Sphingobacterium

 53 sp. and A. gracilis+Sphingobacterium sp. in clean soil, 54 respectively. In these two soils, most of enzyme activities were inhibited in treatments 55 with earthworm alone and earthworm+Sphingobacterium sp.. However, N-related1. Introduction 69Soil is an important source and sink of terrestrial carbon and nitrogen in the 70 biosphere. There are a variety of soil organisms playing an essential role in soil carbon 71 and nitrogen cycle[1,2]. The interactive processes of soil organisms, reconstituting soil 72 carbon stocks and nitrogen cycling in sustainable soil management, have attracted 73 broad attention[3,4]. However, soil metals pollution is becoming an more and more 74 serious environmental and social problem, probably resulting in the changes in the 75 interaction between soil organisms to affect soil carbon and nitrogen cycles[5]. It is of 76 great ecological importance to understand the interactive relationship of soil organisms 77 on the carbon and nitrogen cycle under metal contamination stress.

84

  Nevertheless, currently, previous studies on earthworms in metal contaminated soil 85 have mainly focused on earthworm effects on metal transformation and their 86 ecotoxicological characteristics[10][11][12]. Earthworm effects on soil carbon and nitrogen 87 cycling in metal contaminated soil have not been receiving sufficient attention.88Sphingobacterium sp., a kind of gram-negative bacterium, has a wide range of 89 nutrient sources, high adaptability and wide ecological distribution[16][17][18]. In our 90 previous studies, Sphingobacterium sp. was demonstrated as a kind of dominant 91 microorganism in earthworm gut[25]. Although Sphingobacterium sp. has a strong 92 capacity for organic matter degradation (alkanes and polycyclic aromatic hydrocarbons) 93 to regulate soil carbon and nitrogen transformation in organic polluted soil[19][20][START_REF] Dong | Manipulation of microbial community in the rhizosphere alleviates the 536 replanting issues in Panax ginseng[END_REF][22][23][24], their 94 effect in combination with earthworms on the soil carbon and nitrogen cycle have not 95 yet been clarified, especially under metal pollution stress. 96 Soil C and N cycles invloved a series of biological and biochmical process. Soil 97 carbon and nitrogen fractions, dissolved organic carbon(DOC), alkali-hydrolyzable 98 nitrogen (AN), microbial biomass carbon and nitrogen(MBC and MBN) have been 99 considered as the key indicators to reflect the state of the soil carbon and nitrogen cycles, and their fraction ratios can further provide a more intuitive representation of the overall 101 mineralization or sequestration of carbon and nitrogen in the system. Besides, soil 102 enzymes are metabolic drivers of soil organisms, and their activity can, to some extent, 103reflect not only the changes in the soil microbial community[27], but also the intensity 104 of soil organic matter decomposition and nutrient transformation[28]. Studies on the 105 influence of enzymes on carbon and nitrogen transformation have mostly focused on 106 β-glucosidases (BG) and invertase (INV), which were involved in the decomposition 107 of labile cellulose, sucrose and carbohydrate polymers, and N-acetyl-glucosaminidase 108 (NAG), urease (URE) and proteases (PRO) were involved in the hydrolysis of chitooli-109 gosaccharides, urea and protein, as these enzymes are mainly participating in terminal 110 catalytic reactions or intra-systemic enzymatic reactions, which can reflect the level of 111

113

  effects of earthworm inoculation on soil carbon and nitrogen cycles can indirectly be 114 reflected by studying the changes in soil enzyme activities related to carbon and 115 nitrogen cycles. 116 The aim of this study was to investigate the effects of different species of 117 earthworms (Eisenia fetida and Amynthas gracilis) and exogenous Sphingobacterium 118 sp. on the transformation of soil carbon and nitrogen under clean and Cd-contaminated 119 soil systems. The specific objectives were: (1) to compare and analyse the effects of 120 exogenous Sphingobacterium sp. and two different species of earthworms, individually 121 and simultaneously, on carbon and nitrogen fractions and related enzyme activities in clean and Cd-contaminated soils. (2) to elucidate the role of Sphingobacterium sp. in 123 the participation of earthworms in the carbon and nitrogen cycling process under Cd 124 pollution stress. This study could provide a theoretical basis for an insight into soil 125 carbon and nitrogen cycling processes and biologically driven mechanism, and also 126 provide suggestions for adjusting soil remediation and their further sustainable , earthworm and strain 130 Two representative soils from South China were selected, clean soil (Soil I) and 131 soil contaminated with metals from mining areas (Soil II). Soil I was collected from the 132 tree garden of South China Agricultural University (23°9'N, 113°21'E); Soil II was 133 collected from a paddy field (24°30′N, 113°45′E) about 6 km downstream of the 134 opencast mining area of the copper-sulfur ore, limonite and lead-zinc mine in Dabao 135 Mountain, northern Guangdong Province. The basic physicochemical properties of soil 136 are listed in

177

  carbon and nitrogen (MBC and MBN) were determined via the chloroform fumigation-178

183β

  -glucosidase, protease and N-acetyl-β-D-glucosaminidase activities were all 184 determined by colorimetric assay. Urease, invertase and fluorescein diacetate hydrolytic

  185

187 2 . 4

 24 Statistics analysisOrigin 9.0, and R (Version 3.4.3). T test and variance (ANOVA) analysis were 190 performed. Data was presented as the mean ± SE (standard error). Principal components 191 analysis (PCA) was processed to analyze the relationships of soil carbon and nitrogen 192 fractions and ratios (SOC, DOC, MBC, TN, AN, MBN, DOC:SOC, C:N, AN:TN, 193 MBC:MBN, qMBC, qMBN) and enzymes (BG, INV, URE, NAG, PRO, FDA) in R 194 version 3.4.3 using the "ade4" package. DOC:SOC=DOC/SOC, AN:TN=AN/TN, 195 C:N= SOC/TN, MBC:MBN=MBC/MBN; Microbial quotient carbon (qMBC), 196 qMBC=MBC/SOC; Microbial quotient nitrogen (qMBN), qMBN =MBN/TN.

1

 1 Earthworm survival rate and biomass 199 After 20 d, the survival rate and biomass of both earthworm species decreased 200 (

  207

239

  CSE and CSA treatments. In clean soil, the ratio of AN:TN increased significantly in 240 the treatments involving earthworms and further increased in combination with 241 Sphingobacterium sp. (p < 0.05). MBC:MBN was significantly increased in CS 242 treatment in clean soil and significantly decreased in SS treatment in Cd-contaminated 243 soil (p<0.05). In turn, the ratio significantly decreased in clean soil and increased in 244 contaminated soil when combined Sphingobacterium sp. with earthworms. In clean soil, 245 inoculation with Sphingobacterium sp. and earthworms individually increased qMBC 246 and was highest in CS treatment to control, but decreased in the combined condition. 247 qMBN did not change significantly (p>0.05) in all treatment for both soils.

248 3 . 4

 34 Response of soil enzymes activity to inoculated earthworm and 249 Sphingobacterium sp. 250 The soil enzyme activities were shown in Fig. 3. In clean soil, inoculation of 251 Sphingobacterium sp. alone had no significant effect on enzymes activity, but greater 252 variation in the effect on INV activities. Inoculation of E.fetida individually increased 253 NAG (3.86±0.37 μg•g -

262 3 . 5

 35 Relationships among carbon, nitrogen and related enzymes activity263 with different treatments 264 In clean soils, two principal components (PCs) were identified in the PCA, 265 representing 49.1% of the total variance (Fig. 4a-b). PC1 and PC2 explained 29.6% and 266 19.5% of the total variance, respectively. Significant differences were founded among 267 the treatments, indicating that earthworms and Sphingobacterium sp. had significantly 268 impacts on soil carbon and nitrogen fractions in clean soil (p=0.001). Treatments 269 inoculated with earthworms and both earthworms and Sphingobacterium sp. tended to 270 have low PC1 values, and treatments inoculated with Sphingobacterium sp. alone 271 tended to have low PC2 values. The difference between the treatment C and CSA, CSE 272 was reflected in the latter's significant enhancement of reactive carbon nitrogen such as 273 DOC and AN, while the CE and CA treatments were intermediate between C and CSA, 274 CSE. It can be clearly seen that the difference between C and CS was mainly in the 275 second principal component of MBC, MBC:MBN and qMBC, while CE and CA, CSA and CSE were in mid-way between them. In addition, inoculation with earthworms 277 showed an inhibitory effect on enzyme activity. DOC was negatively correlated with 278 soil enzyme activity.

283

  significantly influenced soil carbon and nitrogen composition in contaminated soils 284 (p=0.017). Treatments inoculated with Sphingobacterium sp. tended to have high PC1 285 values and treatments inoculated with earthworms tended to have high PC2 values. The 286 differences between the S treatment and the earthworm inoculation treatment were 287 mainly reflected in the latter's enhanced effect on SOC, AN and MBN. While the S and 288 SS treatments differed in the latter's enhanced effects on DOC, and the earthworm 289 inoculated treatment was in between. Meanwhile, except for PRO enzyme, earthworm 290 inoculation showed inhibitory effect on enzyme activity.

1

 1 Earthworm growth and survival293Earthworms abundance and biomass decreased after 20 days cultivation in both 294 soils, with significant differences were observed especially in Cd-contaminated soil (p 295 <0.05), consistent with[38]. It might be due to soil nutrient limitation and toxic effects 296

299 4 . 2

 42 Effects of earthworms and Sphingobacterium sp. on soil carbon and 300 nitrogen fractions 301

311

  further significantly increased the reactive carbon and nitrogen content, demonstrating 312 that earthworms and Sphingobacterium sp. were able to stimulate soil carbon and 313 nitrogen cycling, and that there was an intensified effect of the combination. The high 314 microbial biomass carbon content in CS treatment inoculated with Sphingobacterium 315 sp. alone may be due to its adaption to this environment, rapid multiplication and 316 becoming the dominant strain. In contrast, earthworm activities decreased microbial 317 biomass as compared to Sphingobacterium sp. activities alone. It is suggested that 318 earthworms have a role in maintain the ecosystem balance and stabilizing the microbial 319 community structure through feeding[START_REF] Green | Assay for fluorescein diacetate hydrolytic activity: 583 Optimization for soil samples[END_REF] and regulating the allocation of active Therefore, in the treatments of Sphingobacterium sp. in combination with earthworm, 322 the rapid proliferation of Sphingobacterium sp. was prevented. This was further 323 confirmed by the significantly lower qMBC of CSE and CSA compared to CS in Fig.3242e. That is, earthworms reduced the efficiency of soil microorganisms in utilizing 325 carbon sources for conversion into microbial biomass carbon. Additionally, since 326 Sphingobacterium sp. is the dominant gut bacterium of E.fetida and genera of Amythas, 327 the gut can provide a shelter for it to not die completely under Cd-contaminated stress 328[25].329In contaminated soil, Sphingobacterium sp. still showed a facilitation of carbon 330 mineralization (Fig.1a, b; Fig.2a), although the ability to do so was diminished, 331 indicating its ability to mineralize carbon and to tolerate metal contamination stress.332Earthworms, on the other hand, showed carbon sequestration in Cd-contaminated soils 333 with low soil organic carbon content, in contrast to their behavior in clean soils. This is 334 probably due to the biased feeding of organic matter by earthworms to resist metal 335 toxicity, and then, through gut peristalsis and body surface mucus, promote the 336 formation of organic matter-rich aggregate and the protective effect of mucus 337 substances, which reduces carbon loss[6,[START_REF] Schimel | Microbial control over carbon cycling in soil[END_REF][START_REF] Shankar | Saccharification of the pretreated 592 husks of corn, peanut and coffee cherry by the lignocellulolytic enzymes secreted by 127[END_REF][46][47]]. It's also probably explained by 338 contaminated soils containing carbon that is stable and not easily mineralized. In PCA 339 analysis, positive correlation between β-glucosidase and N-acetyl-β-D-340 glucosaminidase activity also indicated that soil microorganisms tend to use the active 341 carbon pool generated by the decomposition of humus formed by litter (Fig.4c; Fig.S1), i.e., favor the accumulation of recalcitrant soil carbon pools. In addition, both 343 dissolved organic carbon and the dissolved organic carbon to soil organic carbon ratio 344 were significantly lower in the SSE treatment than in the other treatments, which may 345 be influenced by the lower survival rate of earthworms and their reduced biological 346 effect capacity in this treatment. For the other carbon and nitrogen fractions, earthworm 347

349 4 . 3

 43 Effect of earthworms and Sphingobacterium sp. on soil enzyme 350 activity 351

  352

3 .

 3 In clean soil, inoculation of earthworms and Sphingobacterium sp. had no significant 353 effect on soil enzyme activities on the whole, but activated enzyme activities in some 354 treatment. In terms of trends, inoculation with earthworms showed weak inhibition of 355 β-glucosidase, urease, and fluorescein diacetate hydrolytic activities, opposite to the 356

4. 4

 4 Effect of earthworms and Sphingobacterium sp. on the balance of soil 387 carbon and nitrogen stabilization and mineralization 388Organisms have strong interaction among themselves in soil[3], earthworm and 389 microorganisms contributed together in this study on the balance of soil carbon and 390 nitrogen stabilization and mineralization. In our study, Sphingobacterium sp. can 391 promote soil carbon and nitrogen mineralization, especially in clean soils (Fig.1-2). It 392 also assist earthworm to further promote carbon and nitrogen mineralization(Fig.4a), 393 and E.fetida showed more efficiencies than A.gracilis. In contrast, the combined effect 394 of them in metal contaminated soils was not significant and exhibited carbon 395 storage(Fig.4b), probably due to the biased feeding of organic matter by earthworms 396 to resist metal toxicity, and the nutrient and metal contaminated stress in the soil not 397 only affects the survival status of earthworms, but also makes it difficult for 398 microorganisms to play its part. Ultimately, the contaminated soil exhibited carbon 399 storage because of the stronger stabilizing effect of earthworms than mineralizing effect 400 of Sphingobacterium sp.. It's also probably owing to mineralizable carbon decreases 401 with time of mine contaminated soils and residue more mineral associated organic 402

  404

  soil carbon and nitrogen cycles can indirectly be assessed by studying the changes in soil enzyme 130 activities related to carbon and nitrogen cycles. 131 Given that Sphingobacterium sp. is the dominant bacterial taxon in the earthworm gut, we 132 hypothesized that Sphingobacterium sp. and earthworm could individually promote soil organic matter 133 decomposition and, when combined, exert reinforcing effects. These effects are negatively impacted by metal stress and influenced by the survival strategies of different earthworm species; Additionally, 135 Sphingobacterium sp. and earthworms may play different roles in this combined process, with 136 Sphingobacterium sp. stimulating microbial activities in the earthworm gut. 137 This study aimed to investigate the effects of different earthworm species (Eisenia fetida and 138 Amynthas gracilis) and exogenous Sphingobacterium sp. on the transformation of soil carbon and 139 nitrogen under non-contaminated and Cd-contaminated stresses. The specific research objectives were 140 (1) to analyze and compare the effects of Sphingobacterium sp. and the two earthworm species, when 141 inoculated alone and in combination, on the carbon and nitrogen forms and related enzyme activities 142 in non-and Cd-contaminated soils. (2) to elucidate the effects of Cd pollution stress on this process 143 and the underline mechanisms. (3) to clarify the interaction and roles of Sphingobacterium sp. and 144 earthworms in these processes. This study can provide a theoretical basis for insight into the combined 145 effects of earthworms and microorganisms on soil carbon and nitrogen cycling processes, the 146 biologically driven mechanisms, and offer suggestions for adjusting soil remediation practices and 147 their further sustainable management. 148

  363

4. 3

 3 The combined effects of earthworm and Sphingobacterium sp. on the balance 364 between C and N stabilization and mineralization 365

  This work was financially supported by the Natural Science Foundation of China (Grant No. 438 41201305), Guangdong provincial Natural Science Foundation (Grant No. 2021A1515011543), 439 National Science and Technology Fundamental Resources Investigation Program of 440 China (2018FY100300), along with National Key Research and Development Program of China 441 (2022YFC3703100). The authors gratefully acknowledge financial support from China Scholarship 442 Council. 443 Reference

Figure captions:Fig. 1 .

 1 Figure captions: Fig. 1. Soil carbon and nitrogen form characteristics.Results are expressed as the mean value ± SE (n = 3). Different lowercase letters indicate sicanificantly differences (P < 0.05) among the treatments.

Fig. 2 .Fig. 3 .Fig. 4 .

 234 Fig. 2.The ratio of soil carbon and nitrogen forms. SOC, soil organic carbon; DOC, dissolved organic matter; TN, total nitrogen; AN, available nitrogen; C:N, soil organic carbon-to-total nitrogen ratio; MBC, microbial biomass carbon; MBN, microbial biomass nitrogen. Results are expressed as the mean value ± SE (n = 3). Different lowercase letters indicate sicanificantly differences (P < 0.05) among the treatments.

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig. 1. Soil carbon and nitrogen form characteristics. Results are expressed as the mean value ± SE (n = 3). Different lowercase letters indicate sicanificantly differences (P < 0.05) among the treatments.

Fig. 4 .

 4 Fig. 4. Principal component analysis (PCA) of the effect of earthworms and Sphingobacterium sp. on soil carbon and nitrogen fractions and enzyme activities in non-contaminated soil in fractal F1, F2 and F3 planes. (A) Correlation circles of carbon and nitrogen fractions with enzymes activities in non-contaminated soil in fractal F1 and

Fig. 5 .Fig. 1 .

 51 Fig. 5. Principal component analysis (PCA) of the effect of earthworms and Sphingobacterium sp. on soil carbon and nitrogen fractions and enzyme activities in Cd-contaminated soil in fractal F1, F2 and F3 planes. (A) Correlation circles of carbon and nitrogen fractions with enzymes activities in Cd-contaminated soil in fractal F1 and F2 planes. (B) Correlation circles of carbon and nitrogen fractions with enzymes activities in Cd-contaminated soil in fractal F2 and F3 planes. (C) Projection of experimental points according to treatments with or without earthworm and Sphingobacterium sp. in Cdcontaminated soil in fractal F1 and F2 planes. (D) Projection of experimental points according to classification with or without strain and worm in Cd-contaminated soil in fractal F2 and F3 planes.

Fig. 2 .

 2 Fig. 2. The ratio of Soil carbon and nitrogen fractions.

Fig. 3 .

 3 Fig. 3. Characterization of soil enzyme activity.

Fig. 4 .

 4 Fig. 4. Principal component analysis (PCA) of the effect of earthworms and Sphingobacterium sp.

(

  a) correlation circles ofcarbon and nitrogen fractions with enzymes in clean soil . (b) Projection of experimental points according to treatments with or without earthworm and Sphingobacterium sp. in clean soil. (c) Correlation circles of carbon and nitrogen fractions with enzymes activities in contaminated soil.

FigureFig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .

 1234 Figure Click here to access/download;Figure;Figures.docx

  (a) correlation circles ofcarbon and nitrogen fractions with enzymes in clean soil . (b) Projection of experimental points according to treatments with or without earthworm and Sphingobacterium sp. in clean soil. (c) Correlation circles of carbon and nitrogen fractions with enzymes activities in contaminated soil. (d)Projection of experimental points according to treatments with or without earthworm and Sphingobacterium sp. in contaminated soil.

combined effects of earthworm and 1 Sphingobacterium sp. on soil C, N forms and enzyme 2 activities in clean and Cd-contaminated soil 3

  Sphingobacterium sp. was able to assist earthworms with improving carbon and nitrogen mineralization in clean soil. In contaminated soil, earthworm alone and earthworm combined with Sphingobacterium sp. both facilitated carbon storage. These findings provided insights into the combined effects of earthworms and microbes on carbon and nitrogen cycling during soil remediation.Li Jia b , Qing Liu a, c , Siyi Chen a , Kexue Liu d , Yiqing Chen a , Mikael Motelica-Heino b , 4Hesen Zhong a , Menghao Zhang a , Cevin Tibihenda a , Patrick Lavelle e , Jun Dai a , Chi Zhang a* 5 a College of Natural Resources and Environment, South China Agricultural University, 6

	7	Keywords:Earthworms; Sphingobacterium sp.; Cd-contaminated soil; carbon and nitrogen fractions; enzyme activities Different Guangzhou, 510642, China
	8	b ISTO UMR 7327 CNRS-Université d'Orléans, Orléans, 45071, France
	9	c Guangzhou Experimental Station of Chinese Academy of Tropical Agricultural Sciences,
	10	Guangzhou, 510850, China
	11	d School of Resources and Planning, Guangzhou Xinhua University, Guangzhou, 510520, China
	12	e IEES-BIODIS/Sorbonne University, Centre IRD, 32 rue Henri Varagnat, Bondy Cedex, 93143,
	13	France
		fetida+Sphingobacterium sp. and A. gracilis+Sphingobacterium sp. in clean soil, respectively. In
		these two soils, most of enzyme activities were inhibited in treatments with earthworm alone and
		earthworm+Sphingobacterium sp.. However, N-related enzyme activities were increased in
		treatments with E. fetida in clean soil and treatments with A. gracilis in contaminated soil(NAG
		activities), protease activities in treatments with Sphingobacterium sp. and treatments with A.

biomass carbon and nitrogen) and C-and N-related enzyme activities (βglucosidase(BG), invertase (INV), urease(URE), N-acetyl-β-D-glucosaminidase(NAG), protease(PRO) and fluorescein diacetate hydrolysis (FDA)), evaluating their interaction on soil C and N cycles. E. fetida and A. gracilis inoculation alone increased DOC contents significantly by 75.8 % and 53.6 % comparing with no earthworm control in clean soil; while they increased AN contents significantly by 32.9% and 20.9% as well, respectively. Inoculation of Sphingobacterium sp. alone promoted microbial biomass carbon reaching 1685±292 mg•kg -1 in clean soil tripled of control. Moreover, the combination of earthworms and Sphingobacterium sp. increased DOC contents by 212% and 134%, and AN contents by 31.3% and 25.4% in treatments of E.

gracilis in contaminated soil(PRO acivities). Our results concluded that earthworm inoculation alone significantly promoted mineralization of carbon and nitrogen in clean soil rather than contaminated soil;

These authors contribute equally to this work: 14

  E. fetida was more effective on C mineralization than A.gracilis.

	29	 Earthworm improved mineralization of C and N in clean soil and C storage in
		contaminated soil.
	31
	32	 Sphingobacterium sp. assisted earthworm with improving C and N
		mineralization in clean soil rather than contaminated soil.
	15	Li JIA
	16	Address: ISTO UMR 7327 CNRS-Université d'Orléans, Orléans 45071, France; Email:
	17	jialila@163.com
	18	Qing LIU
	19	Address: Guangzhou Experimental Station of Chinese Academy of Tropical Agricultural
	20	Sciences, Guangzhou, 510850, China; Email: 178165224@qq.com
	21	Siyi CHEN
	22	Address: College of Natural Resources and Environment, South China Agricultural University,

30



Table 1 .

 1 After 20 days, the earthworms were hand-picked and soil samples were collected

	140
	141	of acclimation in the laboratory, earthworms with similar individual weight and a
		mature clitellum were selected for experiment.

137

Earthworms Eisenia fetida (E. fetida, epigeic specie) and Amynthas gracilis 138 (A.gracilis, epigeic species) were selected. A.gracilis was collected from South China 139 Agricultural University, E. fetida were laboratory-raised earthworms. After two weeks 142 Sphingobacterium sp. was purchased from BeNa Culture Collection (BNCC). 143 Microbial CM0827 Propionibacterium sp. medium consisted of: tryptone 15 g, L-151 soils, with three replicates for each treatment, and 36 pots in total. 152 Soil I: (1) Soil I (C); (2) Soil I + Sphingobacterium sp. (CS); (3) Soil I + E. fetida (CE); 153 (4) Soil I + A. gracilis (CA); (5) Soil I + Sphingobacterium sp. + E. fetida (CSE); (6) 154 Soil I + Sphingobacterium sp. + A.gracilis (CSA).

155 Soil II: (1) Soil II (S); (2) Soil II + Sphingobacterium sp. (SS); (3) Soil II + E. fetida 156 (SE); (4) Soil II + A.gracilis (SA); (5) Soil II + Sphingobacterium sp. + E. fetida ( SSE); 157 (6) Soil II + Sphingobacterium sp. + A. gracilis (SSA). 158 Filled 200 g soil through 2 mm mesh into the pot (caliber x bottom diameter x 159 height: 75 x 53 x 90 mm) with microporous bottom. Stabilized the soil moisture for 48 164 CSA, CSE, SS, SSA and SSE treatments, respectively. To prevent earthworms escape, 165 the top and bottom of each pot were covered with a fine nylon mesh with 1 mm pore diameter. The temperature in the laboratory was controlled at 25°C. 167 173 Soil pH was measured using a glass electrode in a 1:2.5 (w/v) soil/water 174 suspension. Soil clay, and soil organic carbon (SOC), alkali-hydrolyzable nitrogen (AN)

Table 2

 2

	204
	205	Specifically, CE and CSE decreased by 17.4% and 19.4%, respectively, CA and CSA
	206	by 7.68% and 13.7%, respectively; SE and SSE decreased by 17.6% and 77.2%,
		respectively; SA and SSA both by 18.9%.

). In terms of survival rate, only the SSE treatment showed a significant 201 decrease of 68.7% (p<0.05), while the other treatments showed no significant variation.

202

While earthworm biomass decreased by 7.68%-77.2% (p<0.05), the decrease was 203 greater in Cd-contaminated soil than in clean soil, and more in E.fetida than A.gracilis.

  It was reduced by 16.7%, 14.8%, 15.6%, 16.7%, and 15.9% in CS, CE, CA, CSE, CSA

	211
	212	treatments, respectively. Furthermore, DOC increased significantly in the presence of
	213	earthworms (p<0.05), and was highest in the CSE and CSA treatments at 659±19.2
	214	mg•kg -1 and 430±82.8 mg•kg -1 , respectively. Individual inoculation with
	215	Sphingobacterium sp. significantly increased MBC up to 1685±292 mg•kg -1 (p<0.05).
	216	Whereas, in all the treatments inoculated with earthworms, there was no significant
	217	change in MBC content compared to C treatment, but it was significantly decreased
		compared to CS treatment.

. In clean soil, 209 Sphingobacterium sp. and earthworms significantly reduced SOC content (p<0.05). 218 In Cd-contaminated soil, SS treatment reduced the SOC content, decreasing to 219 16.0±0.44 g•kg -1 . while SOC increased in all treatments involving earthworms and was 220 significantly higher than S treatment (16.0±0.44 g•kg -1 ) under combined effect of SSE 221 (17.8±0.28 g•kg -1 ) and SSA (17.5±0.50 g•kg -1 ) (p<0.05). Compared to control, MBC 222 was slightly decreased in SS treatment and the variation was smaller than in CS 223 treatment.

  . In clean soil, the

	225
	226	earthworm activity significantly increased AN content (p<0.05), which increased by
	227	32.9%, 20.9%, 31.3% and 25.4% in CE, CA, CSE and CSA treatments respectively,
	228	while Sphingobacterium sp. did not significantly affect it (p>0.05). However, the
	229	mineralization indexes of nitrogen were not significantly changed in contaminated soil
		(p>0.05), but the trend of AN in all treatments was consistent with in clean soil. In

230

addition, TN and MBN were relatively stable in both soils with no significant changes 231 under the activities of Sphingobacterium sp. and earthworms.

  •h -1 ) activities but decreased URE activities. Moreover, inoculation of A.gracilis individually significantly decreased BG activities (p<0.05). As for the earthworm inoculation treatment, it was inhibited except 260 for SSA, which increased INV, and SA, which increased NAG and PRO activities.

	255
	256	In combination condition, both significantly inhibited BG activities (p<0.05), with no
	257	significant effect on other enzyme activities. In contaminated soil, Sphingobacterium
	258	sp. slight increased BG activities, but there was no significant difference among the
	259	treatments. PRO activity was significantly increased only in SS treatment, reaching
	453±6.64 μg•g -1 •h -1 . 261



  That is, soil carbon and 416 nitrogen index did not show a strong correlation. To reveal the under mechanisms, we 417 need to analyze them in the context of multiple biological and environmental factors. Sphingobacterium sp. could promote C mineralization in both soils. 27  Earthworms facilitated C and N mineralization in non-contaminated soil. 28

	Manuscript File		Click here to view linked References
		431	However, the effect of earthworm and Sphingobacterium sp. on soil nitrogen was no
	26	Highlights
		432	significant in contaminated soil. This study provides a clear understanding on the
		433	different effects of earthworm and microorganisms on soil carbon and nitrogen cycling
	29	 Earthworms enhanced C stabilization in Cd-contaminated soil.
	30	in clean and contaminated soil. In further study, the complex interaction mechanisms  Sphingobacterium sp. could assist earthworms in improving C and N mineralization in non-434
	31	435	of earthworm and microorganisms can be designed and tested by some methods about contaminated soil.
		436	molecular biology or metabolomics. Additionally, the interaction of earthworm and
		437	microorganisms in soil remediation technology could be explored to adjust and rebuild
		438	a sustainable soil management practice.
	12	These authors contribute equally to this work:
	13	Li JIA
	14	Address: ISTO UMR 7327 CNRS-Université d'Orléans, Orléans 45071, France; Email: li.jia@cnrs-orleans.fr
	15	418 440 Qing LIU	This work was financially supported by the Natural Science Foundation of China
	16 17	5. Conclusion (Grant No. 41201305), Guangdong provincial Natural Science Foundation (Grant No. Address: Guangzhou Experimental Station of Chinese Academy of Tropical Agricultural Sciences, Guangzhou, 419 441 510850, China; Email: 178165224@qq.com
	18	This study on soil carbon and nitrogen fractions and enzyme activities to 2021A1515011543), National Science and Technology Fundamental Resources Siyi CHEN 420 442
	19	Address: College of Natural Resources and Environment, South China Agricultural University,
	20	investigate whether Sphingobacterium sp. can assist different species of earthworm in Investigation Program of China (2018FY100300), Guangdong provincial Agricultural Guangzhou 510642, China; Email: 1551749709@qq.com 421 443
	21 22	influencing soil carbon stabilization and nitrogen cycling under clean and Cd-Science and Technology Development and Resources and Environmental Protection *Corresponding author: 422 444 Chi ZHANG
	23	contaminated soil. Overall, earthworm inoculation alone exhibited facilitating carbon Management Project (Grant No. 2022KJ161) along with National Key Research and Address: College of resources and environment, South China Agricultural University, Guangzhou, China, 510642; 423 445
	24	Tel: 0086 13699712963, Email: zhangchi2012@scau.edu.cn
		424 446	and nitrogen mineralization in clean soil, while Sphingobacterium sp. inoculation alone Development Program of China (2022YFC3703100). The authors gratefully
	25	425 447	facilitated carbon and nitrogen mineralization in both soils. In clean soil, earthworm acknowledge financial support from China Scholarship Council.
		426	effect showed species-dependent and E.fetida was more effective than that of A.gracilis.
		427	Sphingobacterium sp. could enhance earthworm effects on carbon mineralization in
		428 448	clean soil. While in cadmium contaminated soil, earthworm and Sphingobacterium sp.
		429 449	combined inoculation improved soil carbon storage rather than carbon mineralization
		450	in treatments with earthworm, but no significant effects between earthworm species.

Table list :

 list Results are expressed as the mean value ± SE (n = 3). The initial letters C and S represents the soil I and soil II. CE, CA, CSE and CSA represents the treatments with E. fetida, A. gracilis, Sphingobacterium sp. + E.fetida and Sphingobacterium sp. + A.gracilis in soil I, while SE, SA, SSE and SSA represents the treatments with E. fetida, A. gracilis, Sphingobacterium sp.+ E. fetida and Sphingobacterium sp. + A. gracilis in soil II, respectively. Different lowercase letters indicate sicanificantly differences (P < 0.05) between different treatments.

	Table2 Survival rate and mean biomass loss of earthworm at the 20th day
	Table1 Properties and Cd concentrations of soil Treatments	Survival rate (%)	Mean biomass loss (%)
		pH	Clay (%) CE	SOC (g•kg -1 ) TN (g•kg -1 ) 95.2 ± 4.76a	C:N 17.4 ± 13.3b Cd (mg•kg -1 ) DTPA-Cd (mg•kg -1 )
	Soil I	4.73 ± 0.05 18.60 ± 3.85 CA	27.0 ± 0.37 86.1 ± 7.35a 1.30 ± 0.05 20.8 ± 0.64 7.68 ± 4.54b 0.13 ± 0.15	0.007 ± 0.001
	Soil II 4.40 ± 0.02 19.42 ± 0.49 CSE	16.6 ± 0.12 91.1 ± 4.49a 1.62 ± 0.05 10.3 ± 0.25 19.4 ± 5.26b 0.64 ± 0.20	0.164 ± 0.000
	Note: Soil I and Soil II represents non-contaminated soil and contaminated soil, respectively. SOC, soil organic carbon; CSA 87.8 ± 6.19a 13.7 ± 10.2b
	TN, total nitrogen; Cd, cadmium. Results are expressed as the mean ± SE (n = 3). SE 77.8 ± 11.1a 17.6 ± 5.22b
			SA		100 ± 0a	18.9 ± 5.54b
			SSE		31.2 ± 6.10b	77.2 ± 3.99a
			SSA		100 ± 0a	18.9 ± 3.27b
	Note:

  Table2The survival rate and biomass of earthworm at the 20th day Results are expressed as the mean value ± SE (n=3). Initial C and S represents the soil I and soil II. CE, CA, CSE and CSA represents the treatments with E.fetida, A.gracilis, Sphingobacterium sp.+ E.fetida and Sphingobacterium sp.+A.gracilis in soil I, while SE, SA, SSE and SSA represents the treatments with E.fetida, A.gracilis, Sphingobacterium sp.+ E.fetida and Sphingobacterium sp.+A.gracilis in soil II, respectively. The same below. Different letters indicate significant differences (p<0.05) between different days.

		Survival rate/%		Biomass /g•pot -1
	Treatments
		Before incubation The 20th day	Before incubation The 20th day
	CE	100±0a	95.2±4.76a	3.02±0.05a	2.49±0.19a
	CA	100±0a	86.1±7.35a	3.05±0.05a	2.81±0.03b
	CSE	100±0a	91.1±4.49a	3.00±0.11a	2.41±0.07b
	CSA	100±0a	87.8±6.19a	3.22±0.06a	2.79±0.24a
	SE	100±0a	77.8 ±11.1a	2.94±0.04a	2.43±0.12b
	SA	100±0a	100±0a	3.10±0.11a	2.52±0.19a
	SSE	100±0a	31.2±6.10b	3.12±0.13a	0.71±0.05b
	SSA	100±0a	100±0a	3.07±0.04a	2.49±0.05b
	Note:

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e IEES-BIODIS/Sorbonne Université, Centre IRD, 32 rue Henri Varagnat, Bondy Cedex, 93143, France

quotient nitrogen (qMBN), qMBN = MBN/TN.

of cadmium-contaminated seleniferous soils, Ecotoxicol. Environ. Saf. 245 (2022) 114122,

Table1Properties and Cd concentrations of soil.

Table2 Survival rate and mean biomass loss of earthworm at the 20th day.

Acknowledgements

439

Acknowledgements

https://doi.org/10.1016/j.jhazmat.2020.124738.

Table list:

Table1 Properties and Cd concentrations of soil.

Table2

The survival rate and biomass of earthworm at the 20th day.

Table (Editable version)

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Declaration of interests

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☒ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Chi ZHANG reports financial support was provided by Guangdong Provincial Natural Science Foundation. Chi ZHANG reports financial support was provided by National Natural Science Foundation of China.