1 Introduction

The atmospheric concentration of carbon dioxide (CO2) has been steadily increased during the last 12 years at the rate of 1.9 μmol mol−1 year−1 and is anticipated to be as high as 550 μmol mol−1 by the middle of the twenty-first century (IPCC 2007). The increased root and shoot biomass by greater photosynthetic assimilation rates under elevated CO2 could lead to a fraction of the additional fixed carbon (C) releasing into the rhizosphere by root exudation (Allard et al. 2006; De Costa et al. 2003a, b, 2006). The soil organic carbon (SOC) contributes to enhancing mineral weathering, nutrient mobilization, and assimilation by the soil microbial biomass, which may play an important role in stimulating microbial and enzyme activity in rhizosphere soils.

Soil contamination by with heavy metals is another widespread and serious issue and is on account of both natural and anthropogenic activities. The presence of toxic metals in soil increases the risk of adverse effects on its health and physicochemical properties. In China, more than 2.0 × 109 ha of land is contaminated with heavy metals (Guo et al. 2011). Among heavy metals, cadmium (Cd) is a heavy metal toxic at very low exposure level that provokes acute and chronic effects on health and environment (NCMWHO2003) and is often present in contaminated soils. It has been widely reported that Cd adversely impact on soil biological functions, including the activity of enzymes (Hassan et al. 2013; Renella et al. 2004) and the diversity of soil microbial community (Pan and Yu 2011; Yang et al. 2019).

It is reported that the carbon imported to soils would be up to 40% through root exudation (Lynch and Whipps 1991), a process that is influenced by plant growth response to elevated CO2, which led to altering the bioavailability and mobility of heavy metals for plants and microorganisms (Wang et al. 2006). As a result, the combination of elevated CO2 and heavy metals would affect the rhizosphere microenvironment of plants. Some studies suggested that elevated CO2 changes the content of SOC and microbial activity in rhizosphere of wheat and pine seedlings exposed to heavy metal pollution (Jia et al. 2014; Kim and Kang 2011). We previously implied, in a short-term pot experiment, elevated CO2 combined with Cd/Pb contamination led to varied soil organic compounds and enzyme activities in rhizosphere of wheat and R. pseudoacacia seedlings (Huang et al. 2016; Jia et al. 2016a), all of which revealed the various responses of rhizosphere microenvironment among different plant species under elevated CO2 plus heavy metal contamination. To our knowledge, many studies have investigated the correlations between the SOC and microbial activities in the rhizosphere microenvironment under stress of elevated CO2 or heavy metals (Khan et al. 2018a, b; Sardar et al. 2007), which much less focus on the interactions of multiple factors. In fact, elevated CO2 and heavy metal pollution generally occur simultaneously with the development of industrialization and urbanization (Sun et al. 2010). Thus, the interactive effects of multiple environmental factors on the correlations between the SOC and microbial activities in the rhizosphere microenvironment should be studied.

Robinia pseudoacacia L. is a promising woody species which can be applied to restoration of degraded ecosystems due to its fast growth, deep root system, and a tolerance of low nutrient levels and heavy metals (Liu et al. 2013; Vlachodimos et al. 2013; Yang et al. 2015). On account of these advantages, R. pseudoacacia is frequently used for phytoremediation in heavy metal-polluted soils (Yang et al. 2015). It is thus significant to investigate the effects of elevated CO2 and Cd pollution on SOC and their relationship to microbial properties in the rhizosphere of this plant species.

Therefore, the main aims of this study were to explore the interactive effect of rising atmospheric CO2 concentration and Cd contamination on soil organic carbon fractions and microbial properties in rhizosphere soils of Robinia pseudoacacia L. seedlings. To address this issue, we designed an open-top field chamber experiment with four treatments: control, elevated CO2, Cd treatment, and elevated CO2 + Cd. And we measured SOC concentration (TOC, DOC, and MBC) and soil microbial activities (soil microorganisms and enzyme activities) in the rhizosphere soils of Robinia pseudoacacia L. seedlings. We hypothesized that (1) Cd treatment would inhibit SOC concentration and soil microbial activities. (2) The negative effects of Cd treatment would be essentially offset by the elevated CO2 condition. (3) Elevated CO2 would stimulate the correlation between SOC and microbial activity under Cd treatment.

2 Materials and Methods

2.1 Soils Preparation and Plant Species

The experimental soils were collected from the surface layer (0–20 cm) of cultivated land in Central Shaanxi, China (34°16′N, 108°54′E). The type and basic chemical characteristics of soil are shown in Table 1. Fresh soils were passed through a 5-mm sieve and pretreated with Cd using a dissolved solution of CdCl2·2H2O. Three levels of Cd were selected to contamination soils on the basis of current environmental quality standard GB 15168-2018 in China: 0.2 (the control, Cd0, with no added Cd), 1.2 (Cd1), and 5.2 mg Cd kg−1 (Cd5) dry soils.

Table 1 Type and basic chemical characteristics of soil used in this study
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Robinia pseudoacacia L. seeds were supplied by Northwest A&F University, China. “A&F” is the abbreviation for the “Agriculture and Forestry”.

2.2 Experimental Site and CO2 Concentration

The experiment site was located on the Weishui Campus of Chang’an University, Xi’an, China (34°15′N, 108°55′E) from June to September 2014. The climate was characterized by an annual precipitation fluctuates between 508 and 702 mm and an average temperature of 13.6 °C (1995–2010). Three levels of CO2 concentration were adjusted by an automatic control system which can regulate the influx rate of CO2 or air: 385 ± 19 μmol mol−1 (the control, ACO2), 500 ± 20 μmol mol−1 (E1CO2), and 700 ± 23 μmol mol−1 (E2CO2), respectively. All treatments were arranged in a randomized block design with three replicates to test for homogeneity of treatments. Three hexagonal OTCs (4.4 m dia × 1.6 m tall) were established under ACO2 (the control), three chambers were maintained under E1CO2, and the other three chambers were established under E2CO2. These chambers kept similar light exposure and microhabitat characteristics. The humidity, temperature, and soil water content were measured automatically every 60 s, and air temperature was noted at every 10 min during experiment period. Average temperature and humidity in the ACO2, E1CO2, and E2CO2 chambers throughout the experiment were 30.5 °C (77.1%), 30.0 °C (76.6%), and 29.9 °C (76.3%), respectively. The effects of temperature and humidity were negligible as the air temperature and humidity were almost identical across all chambers.

2.3 Pot Experiment

Experiment was conducted in plastic pots (70 cm long × 50 cm tall × 40 cm wide) and each pot contained 25 g soils (a control with no Cd was established). Each treatment was prepared with three replicates. On 5 June, Robinia pseudoacacia L. seeds were planted in each pot to obtain 40 seedlings per pot after emergence. The pots were then placed in the open-top chambers. Soil water content was maintained at 60 ± 2.3% of field capacity by watering as needed and was measured with a hand-held probe (IMKO, Germany) throughout the experiment period to exclude influence of soil moisture. The treatments consisted of (1) ACO2 + Cd0, Cd1, Cd5; (2) E1CO2 + Cd0, Cd1, Cd5; (3) E2CO2 + Cd0, Cd1, Cd5. The measurements of these parameters were performed in triplicate. Weeds and litter were monitored and removed from pots by hand to reduce their impact on seedling growth.

2.4 Rhizosphere Sampling

According to the method described by Jia et al. (2014), rhizosphere samples were collected in July, August, and September. Three samples were randomly extracted from each pot. These three soil samples from each pot were mixed to obtain one composite sample. Each composite sample was passed through a 2-mm mesh to remove visible living plant material and divided into two subsamples. One was air-dried to determine soil organic carbon fractions, and the other was stored at 4 °C prior to microbial analysis.

2.5 Soil Organic Carbon Fractions

Soil total organic carbon (TOC) was determined by the K2Cr2O7-H2SO4 oxidation method as described by Nelson and Sommers (1982). Dissolved organic carbon (DOC) was measured by adding soil to water at a ratio of 1:10 (w/v) and assayed with TOC analyzer (TOC-5050A, Shimadzu, Japan). Microbial biomass carbon (MBC) was assayed by fumigation extraction method according to Vance et al. (1987). The measurements of these parameters were performed in triplicate.

2.6 Soil Microbial Analysis

The numbers of bacteria, fungi, and actinomycetes were determined by colony forming units (CFU) using modified plate-dilution technique which was based on meat peptone agar, Thayer-Martin agar, and Gause’s starch agar, respectively (Yang et al. 2009). Five soil enzyme activities were assayed: for urease activity, the release of NH+ was assayed during the hydrolysis of urea (as a substrate) in Tris buffer (reported as mg NH4-N h−1 g dry soil equivalent−1) (Tabatabai and Bremmer 1972). For dehydrogenase activity, the reduction of 2,3,5-triphenyl tetrazolium chloride to triphenylformazan (TPF) was measured after soil incubated at 24 h at 30 °C (reported as μg TPF h−1 g dry soil equivalent−1) (Casida et al. 1964). For invertase activity, 5 g soil was incubated at 37 °C for 24 h with 15 mL of 8% (m/v) sucrose. The suspension was reacted with 3, 5-dinitrosalicylic acid for colorimetric assay, and absorbance was read at 508 nm (reported as μg glucose h−1 g dry soil equivalent−1) (Xu and Zheng 1986). For β-glucosidase activity (μg p-nitrophenol day−1 g dry soil equivalent−1), 1 g of air-dried soil was incubated for 1 h with p-nitrophenyl-β-d-glucoside at pH 6.0 and measured by spectrophotometric assay (Eivazi and Tabatabai 1999). l-Asparaginase was measured according to Frankenberger and Tabatabai (1991) and was expressed as (μg ammonia h−1 g dry soil equivalent−1). The measurements of these parameters were performed in triplicate.

2.7 Statistical Analyses

A general linear model and type-II sum of squares were used to analyze the effects of CO2, Cd, and their interactions. Two-way analysis of variance (ANOVA) was used to examine the individual and combined effects of CO2 and Cd on different parameters (soil organic carbon fractions, microbial population, and enzyme activity). Tukey’s multiple comparison post hoc tests were used to assess the significance of differences between treatments for each variable. These statistical tests were performed using SPSS (SPSS Inc., version 24.0). The association between SOC, soil microorganisms, and enzyme activities (subdivided into groups of SOC [TOC, DOC, and MBC], soil microorganisms [bacteria, fungi, and actinomycetes], and enzyme activities [urease, dehydrogenase, invertase, β-glucosidase, and L-asparaginase]) was determined by Procrustes rotation using the “protest” function in “vegan” (Oksanen et al. 2013). The Procrustes rotation is a method for determining the similarity between multivariate datasets (Peres-Neto and Jackson 2001). One-way ANOVAs and pairwise t tests were used to identify significant differences between the Procrustes rotation results. These statistical analyses were carried out in R version 3.0 (R Core Team 2013).

3 Results

3.1 Soil Organic Carbon Fractions in Rhizosphere Soils

Elevated CO2 significantly (p < 0.05) led to an increase in the concentration of TOC, DOC, and MBC by 8.6%, 32.6%, and 13.5% in the rhizosphere soil of Robinia pseudoacacia L. seedlings compared to ACO2, while DOC and MBC presented smaller growth trend in the range of 500–700 μmol mol−1 CO2 than in the range of 385–500 μmol mol−1 CO2 (Fig. 1). Cd significantly (p < 0.05) reduced the DOC and MBC concentration by 30.1% and 24.9% but did not have a significant effect on soil TOC concentration. Compared to Cd only stress, ECO2 + Cd increased the TOC, DOC, and MBC concentration by 1.6%, 21.4%, and 16.1% in the rhizosphere soils, respectively (Fig. 1). With no significant variation in soil TOC content, DOC was higher in August than in other months, while MBC increased over time. In addition, the interaction between of CO2 and Cd on DOC and MBC was significant (Table 2).

Fig. 1
figure 1

Concentration of TOC (total organic carbon), MBC (microbial biomass carbon), and DOC (dissolved organic carbon) in rhizosphere soils of Robinia pseudoacacia L. seedlings under different treatments. (Date are means ± SE; n = 9). Cd0, Cd1, and Cd5 in figures represent 0.0, 1.0, and 5.0 mg Cd was added to per kilogram dry soil, respectively. ACO2, E1CO2, and E2CO2 in table represent 385 ± 19 μmol mol−1, 500 ± 20 μmol mol−1, and 700 ± 23 μmol mol−1 CO2 were set in this study, respectively. Different capital letters indicate a significant difference (p < 0.05) between elevated CO2 and ambient CO2 for the same heavy metal level (within the same period); different lowercase letters indicate significant differences (p < 0.05) between treatments for the same CO2 level (within the same period). (The same below)

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3.2 Microbial Population and Enzyme Activity in Rhizosphere Soils

The population of bacteria, fungi, and actinomycetes increased by 11.6%, 20.9%, and 15.3% significantly (p < 0.05) with increasing CO2 levels, while bacteria and actinomycetes had smaller growth trend in the range of 500–700 μmol mol−1 CO2 than in the range of 385–500 μmol mol−1 CO2 (Fig. 2). Relative to the control, bacteria and actinomycetes were significantly (p < 0.05) decreased by 21.5% and 15.9% with increasing Cd, while fungi did not vary significantly (Fig. 2). Bacteria and actinomycetes increased significantly (p < 0.05) from July to September, while the lowest population of fungi occurred in August. Under ECO2 + Cd, bacteria, fungi, and actinomycetes increased by 8.5%, 13.7%, and 11.2% relative to ACO2 + Cd and decreased compared to ECO2 alone (Fig. 2). Significant interactive effects of CO2 and Cd on bacteria and actinomycetes were observed (Table 2).

Fig. 2
figure 2

Microbial population in rhizosphere soils under different treatments. (Date are means ± SE; n = 9)

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Elevated CO2 caused significantly (p < 0.05) an increase in urease, dehydrogenase, invertase, and β-glucosidase by 20.1%, 15.8%, 11.1%, and 11.9% relative to ambient CO2, irrespective of the decreased l-asparaginase by 19.9%. In addition, urease and invertase showed smaller growth trend in the range of 500–700 μmol mol−1 CO2 than in the range of 385–500 μmol mol−1 CO2 (p < 0.05) (Fig. 3). Except for l-asparaginase, most soil enzyme activities increased from July to September. Under Cd treatment, urease, dehydrogenase, invertase, β-glucosidase, and l-asparaginase activity decreased by 24.4%, 22.0%, 19.5%, 34.0%, and 29.4% with increasing Cd concentration (Fig. 3). Except for l-asparaginase, elevated CO2 plus Cd treatment was associated with significantly higher enzyme activities by 12.5%, 17.3%, 15.1%, and 8.3% compared to ambient CO2 + Cd (Fig. 3). Interactive effects of CO2 and Cd on urease, dehydrogenase, and invertase were significant (Table 2).

Fig. 3
figure 3

Enzyme activity in rhizosphere soils under different treatments. (Date are means ± SE; n = 9)

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3.3 Relationship Between Soil Organic Carbon Fractions and Microbial Properties

In order to evaluate the relationship between soil organic carbon fractions (TOC, DOC, and MBC), soil microorganisms (bacteria, fungi, and actinomycetes), and enzyme activities (urease, dehydrogenase, invertase, β-glucosidase, and l-asparaginase), the Procrustes rotation test was used, and the results are shown in Table 3. We found that the relationship between soil organic carbon fractions and the group of bacteria, fungi, and actinomycetes was strongest (20 out of 27 treatments in 3 months showed a significant correlation and the strongest correlations were found here), followed by that of soil microorganisms and enzyme activities (18 out of 27 treatments showed a significant correlation). Significant correlations between the SOC matrix and the enzyme activities matrix were found in only 12 out of the 27 treatments. Pairwise t tests on the Procrustes rotation results revealed that the SOC group and the group of enzyme activities were more significantly related to the soil microorganisms group (P = 0.006 and P = 0.02, respectively) than the relationship between SOC group and enzyme activities group. Overall, the relationship between the groups of SOC, soil microorganisms, and enzyme activities was more significant under elevated CO2 or Cd alone than under elevated CO2 plus Cd treatments.

Table 2 Summary of the results of factorial analysis (ANOVA) (F values) for the effects of CO2, Cd, and their interactions on TOC (total organic carbon), MBC (microbial biomass carbon), and DOC (dissolved organic carbon), microbial population, and enzyme activity in rhizosphere soils
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Table 3 Correlations between SOC (soil organic carbon fractions), Mic. (microbial population), and Enz. (enzyme activity) as derived from the Procrustes test
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4 Discussion

4.1 Soil Organic Carbon Fractions in Rhizosphere Soils

Our results demonstrated that elevated CO2 alone corresponded with increased TOC, DOC, and MBC concentrations (Fig. 1). Uselman et al. (2000) and Jia et al. (2016b) reported that the growth, photosynthesis, and root biomass of Robinia pseudoacacia L. can be enhanced by elevated CO2. This might lead to increased net primary production to the root system, which may explain why the soil organic carbon fractions were higher under elevated CO2 conditions. Similar to our results, Wang et al. (2017) suggested that elevated CO2 probably MBC and DOC content, which was on account of the increased root carbon exudation driven by CO2 as indicated in the literature (Chen et al. 2012; Klamer et al. 2002; Koyama et al. 2019). The content of soil organic carbon fractions has been found to be either increased (Deng et al. 2016; Luo et al. 2006), decreased (Carney et al. 2007; Langley et al. 2009), or unaffected (Keiluweit et al. 2015; Koyama et al. 2018) by CO2 enrichment. Additionally, the smaller growth trend of DOC and MBC concentration in the range of 500–700 μmol mol−1 CO2 than in the range of 385–500 μmol mol−1 CO2 suggested that the decreased growth rate of soil organic carbon content under higher CO2 levels.

Kuzyakov (2001) and Walker et al. (2003) suggested that aboveground litter, root residues, and root exudates should be the main source of soil organic carbon fractions in rhizosphere soils. Because weeds and litter were removed from the experiment pots during our study, the variation in organic carbon in rhizosphere soils could be related to the effect of Cd on Robinia pseudoacacia L. growth. Soil microbial biomass, which plays an important role in nutrient cycling and ecosystem sustainability, has been found to be sensitive to increased heavy metal concentrations in soils (Giller et al. 1998; Vig et al. 2003). As hypothesized, Cd addition decreased DOC and MBC in rhizosphere soils, which could be due to the microorganisms under Cd stress diverting energy from growth to cell maintenance functions (Killham 1985). As hypothesized, we observed that elevated CO2 results in higher DOC and MBC in the rhizosphere of Robinia pseudoacacia L. seedlings grown in Cd-contaminated soils. The threat of heavy metals to plants grown in Cd-contaminated soils can be relieved by elevated CO2 through stimulating shoot and root growth, and overall biomass production (Kim and Kang 2011; Li et al. 2012). The increase in growth and biomass production under elevated CO2 favored organic compound diffusion from roots into rhizosphere soils, leading to increased DOC and MBC content in Cd-polluted soils. Similarly, an increase in microbial biomass C under elevated CO2 + Cd (copared to Cd stress only) was demonstrated by Luo et al. (2019). So, elevated CO2 significantly offset the negative impact of Cd addition on soil organic carbon concentration, due to elevated CO2 stimulating plant growth and productivity or altered carbon allocation belowground, mainly as a result of stimulation of photosynthesis (Kassem et al. 2008; Kim and Kang 2011).

4.2 Microbial Properties in Rhizosphere Soils

Microbes need nutrients from decomposing soil organic matter that they can use to construct themselves; therefore, the increased TOC, DOC, and MBC concentrations observed under elevated CO2 contributed to higher microbial population in rhizosphere soils (Fig. 2). The greater microbial activities on account of elevated CO2 was consistent with previous observations in rice and grass soils (Bhattacharyya et al. 2013; Luo et al. 2014). Extracellular enzyme activity is, in general, positively related to microbial activity measured via respiration (Frankenberger and Dick 1983). Kandeler et al. (2006) have showed that elevated CO2 presented directly or indirectly effect on enzyme activities. We found that the microorganism activity increased under elevated CO2 (Fig. 2), resulting in higher enzyme activities examined here (Kools et al. 2005). In addition, changes in soil organic carbon fractions under elevated CO2 can be associated with the variations in microbial biomass and activities in rhizosphere soils (Bhattacharyya et al. 2013; Luo et al. 2014); Compared to 385–500 μmol mol−1 CO2, bacteria, actinomycetes, urease, and invertase presented smaller growth trend in the range of 500–700 μmol mol−1 CO2 (Figs. 2 and 3), which could be associated with the same variation tendency in DOC and MBC (Fig. 1). However, the decreased l-asparaginase with increasing CO2 indicated that elevated CO2 may show different behaviors in their ability to affect different soil enzyme activities. Yuan et al. (2006) also reported the elevated CO2 increased soil urease activity and decreased the β-glucosidase, invertase, acid phosphates, and β-glucosaminidase activities. In addition, Ebersberger et al. (2003) suggested that the stimulation of elevated CO2 in invertase, xylanase, urease, protease, and alkaline phosphatase activities revealed that the larger biomass of microbes was accompanied by an increase in their activity. It is possible that the variation of soil organic carbon composition could lead to a decrease in bacteria and actinomycetes population with increasing Cd levels. Several authors have pointed out that heavy metals can affect microbial biomass and specific microbial groups (D’Aascoli et al. 2006; Gomes et al. 2010; Shen et al. 2005). As hypothesized, we observed bacteria and actinomycetes decreased with increasing Cd levels, which was consistent with previous studies (Jia et al. 2016a; Pan and Yu 2011). Furthermore, we did not find significant variation in fungi population under Cd stress. Previous studies have suggested that fungi are more resistant than bacteria under the long-term heavy metal contamination stress (Fliessbach et al. 1994; Frostegård et al. 1996). Gao et al. (2010) also reported that the order of sensitivity reaction of soils microbe population responding to Cd polution was actinomycetes > bacteria > fungi. We also found that the response of fungi to Cd differed from bacteria and actinomycetes, indicating that heavy metals could present different behaviors in their ability to affect soil microorganisms.

Heavy metals reduce enzyme activity by masking catalytically active groups and interacting with the complex enzyme-substrate, which generates the impact of denaturation toward active proteins of enzymes (Gianfreda et al. 2005; Zaborowska et al. 2006). The lower urease, dehydrogenase, invertase, and β-glucosidase were also consistent with previous studies (Ma et al. 2015; Tripathy et al. 2014), suggesting that soil C and N cycling can be affected by heavy metals. In addition, it has been manifested that heavy metals have an impact on the biosynthesis of enzymes performed by microorganisms. Therefore, a decrease in microbial abundance derived from higher Cd stress (Fig. 2) would lead to adverse effects on enzyme activity in rhizosphere soils.

The increased microbial population under elevated CO2 plus Cd treatment indicated that an increase in CO2 concentration could counteract the negative effects of Cd on microbial abundance in rhizosphere soils. The opposite effects of elevated CO2 and Cd on microorganisms observed here are consistent with previous studies (Chen et al. 2014; Wu et al. 2009). In addition, the adaption of microorganism could lead to selecting specific species that can utilize a broad spectrum of carbon sources via root exudation when a limited amount of carbon is added to the soil through root turnover (Farrar et al. 2003). Thus, we proposed that the increased organic carbon fractions in rhizosphere soils under elevated CO2 + Cd condition (Fig. 1) had a stimulatory effect on soil microbial population on account of the greater substrate availability. Soil enzymes are mainly produced by a diverse group of heterotrophic microbes (Sinsabaugh et al. 2009). Therefore, an increase in enzyme activity (except for l-asparaginase) under elevated CO2 in combination with Cd stress resulted from the stimulation of microbial abundance and production (Fig. 2), suggesting that the stimulatory effect of elevated CO2 might be greater than the inhibitory action derived from Cd contamination, which demonstrated our hypothesis. Similar with our study, Luo et al. (2019) also reported that elevated CO2 strongly alleviated the negative impact on C-degrading enzyme, to promote the β-glucosidase, cellobiohydrolase, polyphenol oxidase, and peroxidase activities and microbial biomass C concentration in soils, indicating elevated CO2 could stimulate the microbial activities and offset the inhibition of Cd toxicity. In generally, our results showed that elevated CO2 significantly increased the microbial population and soil enzyme activity under Cd stress. Our results support the view that elevated CO2 may influence the soil microbial activity (Kim and Kang 2011) and may offset the inhibition of Cd toxicity on soil microbial activity (Luo et al. 2019). Soil enzyme activity was significantly positively correlated with soil microbes (Groffman et al. 2001); the increased microbial populations stimulated by the large quantities of SOC under elevated CO2 contributed to higher soil enzyme activity. In addition, DOC contains macromolecules including acidic groups such as phenolic OH and carboxyl functional groups (Hofrichter and Fakoussa 2001), and these molecules have crucial impact on the solubility, transport, and bioavailability of heavy metals (Kim and Kang 2011). Therefore, the increased DOC concentration under elevated CO2 plus Cd stress (in comparison with Cd stress alone) influenced soil microbial activity by affecting the environmental behavior of Cd.

4.3 Relationship Between Soil Organic Carbon Fractions and Microbial Properties

Soil organic carbon and microbial properties could be stimulated by various environment factors, such as substrate and oxygen availability, soil moisture, temperature, and soil texture (A'Bear et al. 2014; Ma et al. 2016; Maenhout et al. 2018; Xu et al. 2018). To our knowledge, few studies have considered the relationships between the groups of SOC, soil microorganisms, and enzyme activities in the rhizosphere microenvironment under elevated CO2 and Cd stress. In our study, the Procrustes rotation revealed that the soil organic carbon group and the soil enzyme activities group were more significantly related to the soil microorganisms group (P = 0.006 and P = 0.02, respectively) than the relationship between SOC group and enzyme activities group. Similar with our results, many studies have demonstrated that soil microbial diversity and community structure are correlated with the changes of soil organic carbon (Cookson et al. 2005; Marschner et al. 2003). Xiao et al. (2015) also found that in the soil microbe composition, soil organic carbon fractions (e.g., DOC and MBC) were positively correlated with microbial populations in the Calamagrostis angustifolia wetland. The variation of soil microbes could affect soil carbon loss and soil CO2 emissions (Allison et al. 2010; Carney et al. 2007; Khalid et al. 2019), further to stimulate carbon sequestration in soil (Six et al. 2006). In the respect of the correlation between soil enzyme activity and microorganisms, the significantly positive correlation between them has demonstrated by Groffman et al. (2001). The population and composition of microbes could change the magnitude and quality of enzyme activity, further to stimulate the soil metabolic processes (Ushio et al. 2010). In addition, previous studies confirmed soil enzyme activities were related with the soil organic carbon fractions (Acosta-Martínez et al. 2007; Brzezińska et al. 2005), which due to the soil enzymes produced by microorganisms and play key parts in SOC mineralization process (Ahn et al. 2009). Summing up the above, previous studies have reported the relationships between the SOC, soil microorganisms, and enzyme activity. Soil organic carbon inputs used as substrates by microorganisms can be characterized by the abundance and diversity of microbial communities (Kassem et al. 2008; Wang et al. 2008) as well as the variations in enzyme activities (Kandeler et al. 2006), which demonstrated the tight correlation between SOC, soil microbes, and enzyme activity.

In addition, we revealed that the relationships between the groups of SOC, soil microorganisms, and enzyme activities were more significant under elevated CO2 or Cd alone than under elevated CO2 + Cd, which contrasted with our hypothesis. The data obtained here indicated that elevated CO2 might decrease the correlation between soil organic carbon fractions and microbial activities in the rhizosphere of Robinia pseudoacacia L. seedlings in Cd-contaminated soils. A possible explanation is that elevated CO2 would offset the negative effect on the microbial characteristics, to increase the microbial populations and enzyme activities, microbial biomass C in soil, indicating that elevated CO2 can promote the microbial activities and alleviate the inhibition of Cd toxicity.

5 Conclusion

Both elevated CO2 and Cd contamination could affect soil organic carbon fractions and microbial activities in the rhizosphere microenvironment of Robinia pseudoacacia L. seedling. Our study demonstrated that elevated CO2 offsets the negative effect of Cd on soil microbial activities by stimulating soil microorganisms and enzyme activities. We found that the relationship between soil organic carbon fractions and soil microbial activities were higher under elevated CO2 alone than under elevated CO2 plus Cd, revealing that elevated CO2 could stimulate soil fertility and microecological cycle in the rhizosphere microenvironment exposed to heavy metal.