Introduction

Biochar is a carbon-rich product that is produced via pyrolysis of a wide range of biomass sources (Lehmann et al. 2006). It has been well documented that biochar application to soils brings a number of benefits, including improving soil water storage (Reverchon et al. 2015; Wang et al. 2016), liming effect (Van Zwieten et al. 2010b) and reducing nutrient leaching (Bai et al. 2015; Biederman and Harpole 2013). The improved soil quality by biochar addtion is expected to increase plant growth (Jones et al. 2012; Rajkovich et al. 2012).

However, biochar has a limited capacity in improving nitrogen (N) availability for plant uptake (Biederman and Harpole 2013; Bruun et al. 2012; Nelson et al. 2011). Hence, there is a broad spectrum of plant growth responses to biochar additon, including positive, neutral and negative results (Biederman and Harpole 2013; Kammann et al. 2015; Rajkovich et al. 2012). Previous studies have shown that positive effects of biochar application on plant growth usually appeared in the following cases: (1) biochar produced from specific feedstocks (e.g. manure) has relatively high contents of N (Hass et al. 2012; José and Knicker 2011; Nguyen et al. 2017); (2) the combination of biochar amendment with N fertilizers (Kammann et al. 2015; Nguyen et al. 2017), or (3) N-sufficient soils, mostly are agricultural soils (Olmo et al. 2014). However, it has also been reported that different plant species show different responses to the same biochar addition (Biederman and Harpole 2013; Van Zwieten et al. 2010a). Moreover, some plants can even maintain growth stimulation under N-deficient conditions following biochar application (Jeffery et al. 2011; Liu et al. 2013). N availability following biochar addition is commonly invoked to explain these diverse responses (Biederman and Harpole 2013; Bruun et al. 2012; Nelson et al. 2011; Rajkovich et al. 2012).

Similarly, N availability was found to affect the responses of plant biomass to elevated CO2 in massive body of research, and has been ascribed as the primary factor for the diverse plant growth responses to elevated CO2 (McMurtrie et al. 2008; Oren et al. 2001). Therefore, we hypothesized that biochar, with negligible amounts of N, would induce the same plant responses as elevated CO2 in the N-poor soil. If so, this probably would provide insights to elucidate the diverse responses of plant growth to biochar amendment from the perspective of plant physiology.

Moreover, in the context of steadily rising atmospheric CO2 concentrations (Stocker et al. 2013), it is necessary to evaluate if elevated CO2 concentration would influence the potential of biochar application in improving plant growth. Since elevated CO2 would also increase plant demand for N (Finzi et al. 2006; Langley and Megonigal 2010), it is highly likely that under N-limited conditions, the disadvantages of biochar application in improving N availability would be exacerbated by elevated atmospheric CO2. The CO2-enrichment studies have shown that limited N availability would constrain plant production (Elser et al. 2007; LeBauer and Treseder 2008). Furthermore, if N availability is lower than the N demand, plants would increase their loss of carbon (C) through root turnover, respiration or exudation (Drigo et al. 2007, 2008; Hungate et al. 1997). Therefore, if our hypothesis that biochar addition would induce the same responses as elevated CO2 under N-limited conditions is tenable, elevated CO2 would probably deprive the potential of biochar application in improving plant biomass.

Hence, the objectives of the current study were to (1) investigate if biochar with low level of N would induce the same plant responses as elevated CO2 under N-deficient conditions; and (2) evaluate the influence of elevated CO2 on the potential of biochar addition in improving plant growth. To achieve these goals, we grew three Australian indigenous plant species adapted to harsh environmental conditions, including Austrostipa ramossissima (A. ramossissima), Dichelachne micrantha (D. micrantha) and Isolepis nodosa (I. nodosa) (Table 1), with application of three types of biochar in the N-depleted mine spoil under both ambient (400 μL L−1) and elevated (700 μL L−1) CO2 atmospheric concentrations. We assessed the effects of biochar on C and N allocation at the whole-plant level to elucidate the physiological implications. To avoid the N supply from biochar itself, we used N-depleted biochar in the current study with N content of ≤ 0.1% (Table S1).

Table 1 List of the three plant species used in the current study
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Material and methods

Mine spoil sampling and biochar production

The sampling site of mine spoil used in the current study was Mount Owen Coal Mine Complex (Thiess Pty Ltd), located in the central Hunter valley, 20 km northwest of Singleton, New South Wales (NSW), Australia. The mine spoil was composed of coarse rocks that were not suitable for plant growth because of deficiency of water and essential nutrients, extreme pH, up to 9.7, coarse texture and compacted structure (Fisher 2010).

We divided the sampling site into 40 m × 40 m subplots in September 2013 and collected 1 tonne of spoil from each subplot. Then we transported the mine spoil samples in a cooling truck to the laboratory at the Hawkesbury Institute for the Environment (Western Sydney University, Penrith, NSW, Australia). The material (10 tons) was sieved (104 mm mesh), homogenized and stored at 4 °C until use (within 1 week after sampling).

Biochar was produced from pinewood (Pinus radiata) through slow pyrolysis at three maximum temperatures 650 °C (B650), 750 °C (B750) and 850 °C (B850). Specifically, the pinewood sawdust with a particle size of 25 mm was fed into the reactor continuously at 180 kg h−1, with a rotation of 2 rpm, for 8 h per temperature. This allowed for a biomass residence time of 25 min. The biochar produced was released via a jacketed cooling screw.

Establishment of plants

We firstly sterilized the seeds of A. ramossissima, D. micrantha and I. nodosa (Greening Australia, Richmond, NSW, Australia) with 70% alcohol and 6% sodium hypochlorite and germinated them on sterilized glass beads (3 mm diameter) in a growth chamber set at 25 °C light (16 h) and 15 °C dark (8 h). Uniform 24-week-old seedlings (plumule length 10 cm) were selected and transferred to plastic containers (three seedlings per container) upon detection. The three types of biochar were thoroughly mixed with the mine spoil in the plastic containers (750 cm3) at the rate of 8% by dry weight, totally ~ 6 kg in each plastic container and the moisture kept at ~ 10% (based on dry weight) using demineralized water. The control pots received the same weight of mine spoil (~ 6 kg) with no biochar addition. Within each chamber, all pots were shuffled after each watering period to reduce potential position effects within the growth chambers.

CO2 controlled cabinets

The experiment consisted of four environmentally controlled CO2 flow cabinets (4 m × 5 m × 3 m; Climatic Chambers, Vancouver, Canada), with two cabinets CO2 exposed to elevated CO2 at 700 μL L−1, and the remaining two at 400 μL L−1. An airtight system (13,000 L airtight units) integrated with mass flow controllers (Brooks Smart, DMFC, Emmerson process Management, Pittsburg, PA, USA) in each cabinet enabled the maintenance of a constant atmospheric CO2 level of 400 (ambient) or 700 (elevated) μL L−1. A board infrared gas analyser (IRGA, CARBOCAP, GMT222, Dual Wavelength NDIRsensor, Vaisala, Oyj, Finland) was fitted in each of the flow cabinets to control the injection of scrubbed, pure CO2 gas. The elevated CO2 concentration (700 μL L−1) was obtained by injection of CO2 from a pressurized cylinder and the ambient (400 μL L−1) by removal of CO2 from the air by a solid carbon soda filter (Sofnoline, Sigma, St Louis, MO, USA). Inside each CO2 flow cabinet, the maximum and minimum daily temperatures were 26 and 16 °C, respectively, a relative humidity was 70%. Light intensity was averagely kept at 250 μE. Climate data for the cabinets were stored digitally during the entire incubation period (3 years).

Experimental design

The experiment was set-up according to a split-plot design. In this design, we considered the CO2 flow cabinets as whole plot, while the plant species (Table 1) and four types of biochar treatments (without biochar addition (B0), B650, B750 and B850) were considered as sub-treatments.

Sample collection and analysis

A. ramossissima, D. micrantha and I. nodosa were harvested at 3 years after germination, when plants were dominated by shoot growth. At harvesting, plant biomass was divided into different plant components, including leaves and roots, and roots were shaken gently to remove loosely adhering soil. Plant samples were dried at 65 °C to a constant weight, and the dry weights of leaves and roots were measured. We definded the N-use-efficiency (NUE) as the weight (g) of plant biomass produced per N (g). That is, the inverse of the N concentration in the biomass (Berendse and Aerts 1987). The oven-dried samples were ground to fine powder by a RocklabsTM ring grinder. Approximately 8 mg of the ground root or leaf samples was then transferred to 5 mm × 8 mm tin capsules. The powders were then assessed for total C (TC), total N (TN), and C and N isotope compositions (δ13C and δ15N, respectively) using a Eurovector Elemental Analyser (Isoprime-EuroEA 3000, Milan, Italy). Stable isotope ratios were expressed in conventional δ notation as:

$$ {\updelta}^{13}{\mathrm{C}}_{\mathrm{sample}}\ \mathrm{or}\ {\updelta}^{15}{\mathrm{N}}_{\mathrm{sample}}=\left(\left({\mathrm{R}}_{\mathrm{sample}}-{\mathrm{R}}_{\mathrm{std}}\right)/{\mathrm{R}}_{\mathrm{std}}\right) $$

where δ13Csample or δ15Nsample was the sample of interest, Rsample was its 13C/12C or 15N/14N ratio, Rstd was the 13C/12C or 15N/14N ratio of standard, specifically, PDB (Pee Dee Belemnite) standard for δ13C and the atmospheric air standard for δ15N. The results for δ13C and δ15N were reported as parts per thousand (‰) deviations from the PDB standard and atmospheric air, respectively. The analysis of TC, TN, δ13C and δ15N for biochar was conducted in the same way as biomass samples (Table S1). Biochar physical properties were determined by the laboratory of School of Engineering, University of Western Australia (Wang et al. 2016).

Statistical analysis

We assessed biochar effects under ambient and elevated CO2 concentrations separately. A one-way ANOVA was conducted to determine if plant properties measured in the current study were significantly different under the four biochar treatments (B0, B650, B750 and B850) (n = 4). We used Shapiro-Wilk test to assess whether the data were normally distributed (P > 0.05). The homogeneity of variances was assessed by Levene’s test for equality of variances (P > 0.05). If homogeneity of variances was met, we kept the results of the standard one-way ANOVA. When homogeneity of variances was violated, we carried out the analysis with a one-way Welch ANOVA. The mean values were given along with the standard error for replicate measurements. Means and standard errors were based on the four replicates for each biochar treatment. Tukey test (P < 0.05) was used to determine the significance of differences among the treatments. All analyses were performed using SPSS 22.

Results

The effects of biochar addition on plant C and N

Leaf TN concentrations significantly decreased following the addition of all the three types of biochar for species D. micrantha under ambient CO2 and I. nodosa under both ambient and elevated CO2, where significant increases in leaf total C content (TCweight) were also detected (Tables 2 and 3; Fig. 1b, c). In contrast, no significant alteration in leaf TN concentrations was observed for A. ramossissima under both CO2 concentrations or D. micrantha under elevated CO2, accompanied by the absence of increase in leaf TCweight (Tables 2 and 3; Fig. 1a, b). A. ramossissima under elevated CO2 even exhibited reduction in leaf TCweight, especially significant with the application of biochar produced at 650 and 850 °C (Fig. 1a). On the other hand, biochar addition exerted negligible changes in root TN concentrations across the three plant species and the CO2 treatments, except for D. micrantha that the application of biochar produced at 750 °C significantly increased root TN concentration (Tables 2 and 3).

Table 2 Leaf and root total nitrogen concentration (TN), stable nitrogen isotope ratio (δ15N) and total nitrogen content (TNWeight) of A. ramossissima, D. micrantha and I. nodosa under different biochar treatments exposed to ambient CO2 concentration (400 μL L−1)
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Table 3 Leaf and root total nitrogen concentration (TN), stable nitrogen isotope ratio (δ15N) and total nitrogen content (TNWeight) of A. ramossissima, D. micrantha and I. nodosa under different biochar treatments exposed to elevated CO2 concentration (700 μL L−1)
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Fig. 1
figure 1

Effects of biochar addition on leaf total carbon content (TC (g)) of a A. ramossissima, b D. micrantha and c I. nodosa under ambient (400 μL L−1) and elevated (700 μL L−1) CO2 concentrations. Values are mean + SE (n = 4). B0, without biochar addition; B650, B750 and B850: biochar produced via slow pyrolysis at 650, 750 and 850 °C, respectively. Different lower case letters above the bars indicated significant difference at P < 0.05

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Biochar addition increased root total N content (TNweight) for species D. micrantha and I. nodosa under both CO2 treatments, although the significances varied with biochar pyrolysis temperature. In contrast, the presence of biochar exhibited no significant influence on leaf TNweight (Tables 2 and 3). In contrast, A. ramossissima showed no significant changes in either leaf or root TNweight following biochar amendment under both CO2 concentrations (Tables 2 and 3). Significant improvements in NUE following biochar addition were detected across the plant species and CO2 treatments, except for A. ramossissima under elevated CO2 concentration and D. micrantha with application of biochar produced at 750 °C under both CO2 treatments not statistically significant (Fig. 2). Biochar addition decreased leaf δ15N values across the plant species and CO2 treatments, except for I. nodosa and A. ramossissima under ambient CO2, although the significances varied with biochar pyrolysis temperature (Tables 2 and 3). However, there were no significant effects of biochar application on root δ15N values across plant species and CO2 treatments, except for the application of biochar produced at 650 °C significantly decreasing root δ15N value for I. nodosa under elevated CO2 (Tables 2 and 3). While biochar addition significantly decreased leaf δ13C values for I. nodosa under both CO2 concentrations, no significant changes appeared in leaf δ13C for A. ramossissima and D. micrantha under both CO2 treatments (Fig. 3).

Fig. 2
figure 2

Effects of biochar addition on nitrogen-use-efficiency (NUE) of a A. ramossissima, b D. micrantha and c I. nodosa under ambient (400 μL L−1) and elevated (700 μL L−1) CO2 concentrations. Values are mean + SE (n = 4). B0, without biochar addition; B650, B750 and B850: biochar produced via slow pyrolysis at 650, 750 and 850 °C, respectively. Different lower case letters above the bars indicated significant difference at P < 0.05

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Fig. 3
figure 3

Effects of biochar addition on leaf stable carbon isotope ratio (δ13C) of a A. ramossissima, b D. micrantha and c I. nodosa under ambient (400 μL L−1) and elevated (700 μL L−1) CO2 concentrations. Values are mean + SE (n = 4). B0, without biochar addition; B650, B750 and B850: biochar produced via slow pyrolysis at 650, 750 and 850 °C, respectively. Different lower case letters above the bars indicated significant difference at P < 0.05

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The effects of biochar addition on plant growth

Biochar amendment significantly increased leaf biomass for I. nodosa under both ambient and elevated CO2, whereas biochar addition exhibited no significant effects on leaf biomass for D. micrantha under both CO2 treatments (Table 4). Moreover, biochar application even significantly decreased leaf biomass for A. ramossissima under elevated CO2 (Table 4). For root biomass, significant improvements were observed for I. nodosa under elevated CO2 but only statistically significant for I. nodosa and D. micrantha with application of biochar produced at 650 °C under ambient CO2 (Table 4). Total biomass showed significant improvement across the plant species and CO2 treatments, except for A. ramossissima and D. micrantha under elevated CO2 concentration, although the significances varied with biochar pyrolysis temperature (Fig. 4).

Table 4 Leaf and root biomass of A. ramossissima, D. micrantha and I. nodosa under different biochar treatments in combination with ambient (400 μL L−1) and elevated (700 μL L−1) CO2 concentrations
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Fig. 4
figure 4

Effects of biochar addition on plant total biomass of a A. ramossissima, b D. micrantha and c I. nodosa under ambient (400 μL L−1) and elevated (700 μL L−1) CO2 concentrations. Values are mean + SE (n = 4). B0, without biochar addition; B650, B750 and B850: biochar produced via slow pyrolysis at 650, 750 and 850 °C, respectively. Different lower case letters above the bars indicated significant difference at P < 0.05

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Discussion

Biochar addition induced the same plant responses as elevated CO2

Our results showed that biochar addition significantly decreased leaf TN concentrations for species D. micrantha under ambient CO2 and I. nodosa under both ambient and elevated CO2 (Tables 2 and 3). There are few studies that reported decreases in leaf TN concentrations following biochar addition (Kammann et al. 2011; Lehmann et al. 2003; Noguera et al. 2010; Rondon et al. 2007), which have been ascribed to adsorption or immobilization of N induced by biochar incorporation (O’Toole et al. 2013; Rajkovich et al. 2012). Decreases in leaf TN concentration are commonly seen under elevated CO2 and the dilution due to accumulation of non-structural carbohydrates (NSC) has been proposed as the primary reason (Gifford et al. 2000; Taub and Wang 2008). Interestingly, the corresponding significant increases in leaf TCweight in these cases suggested that dilution of NSC provides a likely explanation for the significant decrease in leaf TN following biochar addition (Fig. 1). This dilution mechanism is further confirmed by the concurrence of the absences of significant decrease in leaf TN and increase in leaf TCweight of A. ramossissima under both CO2 concentrations and D. micrantha when exposed to elevated CO2 (Tables 2 and 3; Fig. 1).

However, it has been widely proved that dilution is not solely responsible for decreasing leaf TN cencentration under elevated CO2 (Poorter et al. 1997; Taub and Wang 2008). Alternatively, many studies suggested that plants decreased N investment in photosynthetic apparatus, particularly Ribulose 1,5 bisphosphate carboxylase (rubisco) and thus, potentially distributes more N in root, which was involved in obtaining the resources that would be needed most (Hermans et al. 2006). In this way, plants could achieve optimal N distribution and thus obtain greater NUE to meet the higher N demand under elevated CO2 (Cotrufo et al. 1998; Taub and Wang 2008; Wolfe et al. 1998). The significant increase in root TNweight, along with the absence of significant alteration in leaf TNweight of D. micrantha and I. nodosa (Tables 2 and 3), suggested that the shift in N partitioning from leaves to roots in the presence of biochar probably also played a role in reducing leaf N concentration. The exception of A. ramossissima (Tables 2 and 3) probably suggested the relatively lower potential for improving NUE. The near-ubiquitous NUE improvement in this study (Fig. 2) lent further qualified support to this conclusion, which also offered a possibility for the plant total biomass enhancement (Fig. 4) in spite of the N deficiencies of biochar and mine spoil used in this study (Fisher 2010). Therefore, our results at least partially explained why some plants could even maintain growth stimulation under N-deficient conditions following biochar application (Jeffery et al. 2011; Liu et al. 2013).

Further evidence in support of the hypothesis that biochar addition induced closely resembled plant responses to elevated CO2 comes from leaf δ15N values. Significant depletion in plant δ15N values under elevated CO2 has been observed in many previous studies (BassiriRad et al. 2003; Billings et al. 2002). This was primarily attributed to increased plant reliance on mycorrhizal fungi for N uptake with the rising of CO2 since substantial N isotopic fractionation occurred during transferring from mycorrihizal fungi to plant (BassiriRad et al. 2003; Garten et al. 2011; Polley et al. 2015). Our data showed significant decreases in leaf δ15N of A. ramossissima and D. micrantha following biochar addition, although the decrease in case of A. ramossissima under ambient CO2 was not statistically significant (Tables 2 and 3). Moreover, the absence of significant decrease in leaf δ15N of I. nodosa under ambient CO2 concentration (Table 2) supported our hypothesis in a different perspective in view of the fact that I. nodosa relied on root for N uptake while A. ramossissima and D. micrantha were associated with arbuscular mycorrhizal (AM) fungi (Table 1). However, the significant decrease in leaf δ15N of I. nodosa when exposed to elevated CO2 (Table 3) released a signal of some level of N limitation (Garten et al. 2011). This is not out of expectation since both CO2 enrichment and biochar addition would induce higher N demand.

Another line of evidence derived from our observation that biochar application tended to reduce TN and δ15N of leaves more pronouncedly than those of roots (Tables 2 and 3), which were commonly seen under elevated CO2 (BassiriRad et al. 2003; Cotrufo et al. 1998). Hence, it remains a strong possibility that biochar addition induced the same plant responses as elevated CO2 in the N-deficient environment in this study, which deserves further investigation.

What is the influence of elevated CO2 on the potential of biochar application in improving plant growth?

As expected, when exposed to elevated CO2, A. ramossissima exhibited no significant improvements in plant total biomass in the presence of biochar (Fig. 4a). Additionally, although not statistically significant, root biomass of A. ramossissima even showed some level of decrease in response to biochar addition (Table 4). This suggested that N demand was probably not fully met and thus, A. ramossissima might increase its root turnover, which is consistent with our hypothesis that disadvantages of biochar application in improving N availability would be exacerbated under elevated atmospheric CO2 and thus, elevated CO2 would deprive the potential of biochar application in improving plant biomass. Similarly, while biochar amendment significantly improved total biomass of D. micrantha under ambient CO2 concentration, no significant increase existed under elevated CO2 (Fig. 4b), which further confirmed our hypothesis.

Conversely, biochar addition significantly increased total biomass of I. nodosa under both CO2 concentrations (Fig. 4c). It was interesting to notice that biochar amendment triggered significant decrease in leaf δ13C of I. nodosa (Fig. 3c), which is commonly seen under elevated CO2 and could be partially ascribed to improved photosynthetic capacity (Leakey et al. 2009; Meyers 2014; Schubert and Jahren 2015). This might suggest that I. nodosa would have larger potential for increasing NUE compared to A. ramossissima or D. micrantha and thus, allowed greater improvement in plant photosynthesis. Further support for this view was derived from our observation that I. nodosa exhibited the only significant increase in leaf biomass in response to biochar addition (Table 4). Therefore, the higher potential for improving NUE of I. nodosa made it is possible that biochar addition continued to improve biomass of I. nodosa under elevated CO2. This is probably why different plant species show different responses to the same biochar addition (Biederman and Harpole 2013; Van Zwieten et al. 2010a).

Conclusion

Our results suggested that in the N-depleted mine spoil, the addition biochar with extremely low level of N induced species-specific decreases in leaf total N concentrations, which was commonly seen under elevated CO2. Under N-deficient conditions, plants could decrease N investment in leaf and thus, potentially distribute more N in root, via which plants could achieve optimal N distribution and thus obtain greater N-use-efficiency to sustain plant growth improvement even under N-limited conditions. Hence, the species-specific decreases in leaf total N with biochar addition among the three plant species used in this study probably reflect different potential in improving N-use-efficiency in an increasing order as follows: A. ramossissima < D. micrantha < I. nodosa. This offers an optional explanation for the broad spectrum of plant growth responses to biochar additon from the perspecitve of plant physiology. Biochar addition also decreased leaf δ15N as elevated CO2, which probably due to increasing plant reliance on mycorrihizal fungi for N uptake to adapt to N deficiency. Hence, it remains a strong possibility that biochar addition induced similar plant physiology responses to elevated CO2 in the N-deficient mine spoil. Moreover, since elevated CO2 would also increase plant N demand, the lower potential for improving N-use-efficience of A. ramossissima and D. micrantha probably only allowed significant increase in total biomass in presence of biochar under ambient CO2, whereas the higher N-use-efficience of I. nodosa could sustain the biomass improvement under both CO2 concentrations. Hence, elevated CO2 decreased the ability of biochar addition in improving plant growth.