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Biochar

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Effects of biochar amendment and nitrogen fertilization on soil microbial biomass pools in an Alfisol under rain-fed rice cultivation

  • Segun OladeleEmail author
  • Adebayo Adeyemo
  • Ajoke Adegaiye
  • Moses Awodun
Original Article
  • 79 Downloads

Abstract

Field studies were conducted over 2 years to determine the response of soil microbial biomass pool to biochar and N fertilizer combinations in a rain-fed rice cropping system. Biochar was applied at four doses: 0 t ha−1, 3 t ha−1, 6 t ha−1 and 12 t ha−1 in combination with N fertilizer at four rates: 0 kg ha−1, 30 kg ha−1, 60 kg ha−1 and 90 kg ha−1 to a Typic Paleustalf Alfisol. Soil samples from two depths (0–10 and 10–20 cm) were collected to determine microbial biomass C (MBC), N (MBN), P (MBP), MBC/N ratio, MBC/P ratio, soil CO2 flux, microbial qCO2, cultivable bacterial and fungal abundance. Biochar and N fertilizer combination effects on MBC, MBN and MBP pools were dependent on biochar doses, N fertilizer rates and soil depth. MBC/N and MBC/P ratios were decreased after 2 years. Soil CO2 flux was maximum at post-seeding stage of rice plant, while decreasing trends occurred at active tillering and harvest stage. Increasing doses of biochar irrespective of its combination with N fertilizer rates decreased CO2 flux and microbial qCO2. Combinations of biochar and N fertilizer increased fungal/bacterial ratio and induced a shift to a more fungal-dominated population after 2 years. Our results suggest that combination of biochar doses (3–12 t ha−1) with N fertilizer rates had stimulatory effects on microbial biomass pools and activity with positive implications for organic carbon accumulation, nitrogen (N) and phosphorus (P) retention in tropical soils.

Keywords

Biochar Nitrogen fertilizer Microbial biomass Metabolic quotient Fungal/bacteria ratio 

1 Introduction

Soil micro-biome is highly influential in controlling processes such as organic matter mineralization, decomposition, nutrient cycling and greenhouse gas emissions (GHGs) (Garcia et al. 2002). The importance of soil microbes is further emphasized by the fact that over 80% of soil functioning is regulated by their activity (Jin 2010). Hence, it is imperative to study how different crop and soil management practices affect microbial biomass and activity. Studying the dynamics of soil microbial abundance and activity is expected to provide insights into the response of soil microbiota to anthropogenic impacts and other geologic influence (Maeder et al. 2002). Soil microbial biomass is a useful indicator of microbial activity and soil quality and can as well monitor changes occurring in soil functioning from the addition of various amendments, management practices and soil pollution (Thies and Rillig 2009).

The use of biochar as an amendment strategy for improving soil quality and as a means of efficiently utilizing and recycling wastes has generated lots of interest worldwide (Oladele et al. 2019). Biochar is the product derived from thermal degradation of organic biomass in the partial presence or absence of oxygen and solely intended for use as a soil amendment (Lehmann et al. 2011; Oladele et al. 2019). It has been extensively studied due to its potential in helping to increase carbon sequestration (Lehmann and Rondon 2006), contaminants removal, nutrients and water retention (Yu et al. 2006), soil pH increase (Oladele et al. 2019), enhanced crop productivity (Rondon et al. 2007; Chan et al. 2007) and GHGs mitigation (Lehmann 2007). The properties of biochar, as well as induced changes in soil physico-chemical properties, have been reported to alter soil microbial biomass pool (Gul et al. 2015). These alterations in soil physico-chemical properties can promote microbial biomass abundance and activities directly through the provision of substrates, suitable habitats and niches. Further, biochar feedstock, pyrolysis conditions, quality and rate of application could also affect microbial biomass pool and biodegradation of biochar (Jin 2010). Contrasting and unclear effects of biochar on soil microbial biomass and activity have been reported. For instance, Zackrisson et al. (1996), Wardle et al. (2008), Lehmann et al. (2011) and Zhang et al. (2014) reported increase in pools of microbial biomass C (MBC), N (MBN) and rates of microbial activities, while Deenik et al. (2010) and Dempster et al. (2011) reported decreased soil microbial biomass and activity. In addition, Matsubara et al. (2002), Yamato et al. (2006), Rondon et al. (2007) and Warnock et al. (2007) reported increased abundance and activity of mycorrhizal fungi in biochar-amended soils, while Gaur and Adholeya (2000) and Wallstedt et al. (2002) reported decreased population and activity of mycorrhizal fungi.

Biochar-amended soils have been shown to alter microbial biomass by inducing a shift to a more fungal-dominated population (Lehmann et al. 2011). This induced change has been suggested to be driven by an increase in the soil fungal community rather than by changes in the bacterial community and the preferential advantage of fungi over bacteria in the degradation of lignin (Lehmann et al. 2011). On the other hand, an increase in bacteria population over fungi has also been reported after soil pH increase by biochar addition in acidic soils. For example, Gómez et al. (2014) and Ameloot et al. (2015) reported decrease in fungal-to-bacterial ratio after biochar amendment, while Prayogo et al. (2014) observed no changes in this ratio. Furthermore, the potentials of biochar amendment in reducing CO2–C flux and sequestering carbon in agricultural soils have been documented (Woolf et al. 2010; Zhang et al. 2016). However, in a meta-analysis, He et al. (2016) reported that biochar amendment significantly increased soil cumulative CO2–C flux by 22% but decreased CO2–C emissions by 9% in N-fertilized soils which suggests that CO2–C flux from biochar-amended soils may depend on N fertilization rates. Based on previous studies and meta-analysis, there exists limited literature explaining biochar and inorganic N fertilizer effects on soil microbial population and CO2–C flux particularly from humid tropical cropland.

Documenting studies on soil microbial biomass dynamics under biochar and inorganic N fertilizer addition will provide a better perspective of carbon sequestration, nutrient mineralization pattern and biochar decomposition. Despite the potential influence of biochar and inorganic N fertilizer on soil quality and health, very little is known about their effects on soil microbial biomass pools. Moreover, present knowledge on soil microbial biomass pools as influenced by biochar amendment is mostly limited to temperate latitudes and comparison between biochar types, doses, varying pyrolysis conditions and laboratory studies. Information on biochar and inorganic N fertilizer combination on soil microbial biomass pools at field scale level in tropical latitudes remains scanty. Specifically, the objective of our study was to determine the effect of rice husk biochar and N fertilizer combinations on microbial biomass pools and CO2–C flux from a Typic Paleustalf Alfisol under rain-fed rice cropping system in south-west Nigeria. We hypothesized that application of biochar and N fertilizer will increase microbial biomass pools, induce change in microbial population and decrease CO2–C flux with consequent effect on nutrient cycling, retention and carbon storage in a humid tropical alfisol.

2 Materials and methods

2.1 Experimental site characteristics

Field experiment was conducted at the experimental station of the Federal University of Technology, Department of Crop, Soil and Pest Management, (7°20′N; 5°30′E, altitude—370 m) during the wet seasons of (August–November), 2016 and (August–November), 2017 at Akure, Southwest Nigeria. Topographically, the site is generally flat and soil area falls into large quantity of red laterite and very little of mangrove swamp of humid tropical equator. Climatic information of the study area indicates an equatorial rain forest belts with annual mean temperature of 25.3 °C (Awopegba et al. 2017). Mean annual precipitation at Akure is 1450 mm with most rainfall occurring from May to September. Soil textural class indicates a proportion of sand (68.8%), clay (25.1%) and silts (6.1%) particles (sandy clay loam) classified as Typic Oxic-Paleustalf (USDA Soil Taxonomy). The bulk density of the top 20 cm of soil was 1.52 g cm−3, soil organic matter (SOM) content was 14 g kg−1 of soil and soil pH (H2O) was 4.95 prior to biochar amendment.

2.2 Biochar

Rice husk obtained from a local rice mill in Akure, Ondo State, Nigeria, was used to produce biochar using a biochar reactor. It was pyrolysed at 350–400 °C for 55 min and at a heating rate of 0.90 °C/s, yielding about 51% biochar by mass. The resulting biochar was allowed to cool before field application. Select chemical properties of the biochar were earlier reported in Oladele et al. (2019).

2.3 Experimental design

The experimental crop used in this study was rice (Oryza sativa) sown between August and November, 2016 and 2017, respectively. An improved variety of upland rice seed (N-U-1/FARO 65) was manually sown by dibbling holes at a spacing of 25 cm by 25 cm and thinned to two plants per hole at 2 weeks after planting. The plant population is close to recommended plants density of 160,000 by AfricaRice Centre, IITA (2014). Field experiment was set up in a randomized complete block design, with each of the experimental plot measuring 2 m × 2 m = 4 m2. Treatments consist of four doses of biochar: 0, 3, 6 and 12 t ha−1 and four rates of nitrogen fertilizer (Urea—46% N): 0, 30, 60 and 90 kg ha−1, which were combined together in factorial to form a total of sixteen treatments with three replicates [(i) N0B0 (control) (ii) N0B3 (iii) N0B6 (iv) N0B12 (v) N90B0 (vi) N90B3 (vii) N90B6 (viii) N90B12 (ix) N60B0 (x) N60B3 (xi) N60B6 (xii) N60B12 (xiii) N30B0 (xiv) N30B3 (xv) N30B6 (xvi) N30B12.], where N = nitrogen fertilizer rate (kg ha−1) and B = biochar rate (t ha−1). Rice husk biochar was evenly mixed with the soil and incorporated at specified doses into the top 15 cm. Biochar was applied once and its residual effect monitored over 2 years. Nitrogen fertilizer was applied in three splits evenly throughout rice growth cycle; the first split was broadcast before seeding; the second and third split were applied along the rows of each plot at rice tillering and booting stage, respectively, to minimize N fertilizer losses. Basal phosphate fertilizer was not applied as pre-seeding soil analysis did show that available phosphate level was adequate, while rice husk biochar provided an inorganic form of potassium fertilizer. Field trial management practices, total rainfall and temperature regime during crop growth period were as reported in an earlier study published by Oladele et al. (2019).

2.4 Soil sampling

Soil samples were collected from five points randomly at the depths of 0–10 and 10–20 cm in each experimental plot. The randomly collected samples were homogenized into one sample and divided into two parts. One part of the soil sample was used for chemical analysis (soil pH, electrical conductivity (EC), organic carbon (Corg), total nitrogen (TN), available phosphorus and potassium), and the other part was prepared for microbial analysis. All samples were immediately stored in sealed plastic bags in a cooler and transported to laboratory and stored at 4 °C. All microbiological determinations were conducted within 3 days of sampling. Soil pH and electrical conductivity (EC) were determined on 1:2.5 of soil/water solution using a pH and conductivity meter (McLean 1982; Rayment and Lyons 2011). Available P was determined by Bray-1 method (Bray and Kurtz 1945), Corg by Walkley and Black (1945) method, TN content by the Kjeldahl digestion (Nelson and Sommers 1982) and exchangeable potassium by ammonium acetate method (Black 1965).

2.5 Microbial biomass measurement

Microbial biomass C (MBC) and N (MBN) in soil were determined by fumigation extraction method (Brookes et al. 1985; Wu et al. 1990), and the value of 0.45 was used for both the fraction of biomass carbon mineralized and the fraction of biomass nitrogen mineralized. Soil samples were collected at the rice flowering stage, thoroughly mixed and ground to pass through a 2-mm sieve. Thereafter, the soil moisture was adjusted to about 40% water holding capacity. Twenty grams of dry weight equivalent of soil was fumigated with ethanol-free chloroform for 24 h. Both fumigated and non-fumigated soils were extracted with 80 ml of 0.5 M K2SO4 by shaking for 30 min on a reciprocating shaker at 40 cycles per minute and then filtered (soil/water = 1:4). For microbial biomass P (MBP) analysis, 5 g of field-moist soil was weighed into a crucible and fumigated in a dessicator with 30 ml of alcohol-free chloroform for 5 days. Both fumigated and unfumigated soil samples were shaken with 35 ml Bray’s 1 extracting solution (0.03 M NH4F + 0.025 M HCl) for 10 min and filtered. Correction for adsorption of P during fumigation was made by simultaneously equilibrating unfumigated soil with a series of P containing standard solutions followed by extraction with the Bray-1 solution. The amount of chloroform released P was determined according to the relationship between P added (from standard solutions or microbial lysis) and P extracted by the Bray-1 solution (Oberson et al. 1997). Phosphorus adsorption during equilibrium is described following the equation proposed by Barrow and Shaw (1975) and adapted by Morel et al. (1997). The amount of microbial P was estimated by assuming a kp factor of 0.4 (Brookes et al. 1985).

2.6 Soil respiration

Soil respiration was measured at different intervals during the rice crop cycle using the gas entrapment method by Hutchinson and Mosier (1981) and Sullivan et al. (2010). Basal CO2 flux was determined at three rice cycle stages: (i) post-seeding stage (ii) active tillering stage and (iii) harvest stage after 2 years of biochar amendment. 10 ml solution of 1 M NaOH was dispensed into a vial and placed under a plastic chamber to trap CO2 evolved from soil sampled at two depths (0–10 and 10–20 cm) from all treatments. Another vial containing 10 ml of 1 M NaOH with the lid open to exclude CO2 evolved from the soil served as control to account for the CO2 trapped from the atmosphere. The trapping solutions in the vials were collected after 3 days (72 h) at each rice cycle stages. Vials were covered with airtight seal immediately and taken to the laboratory for analysis. After 30 min, the whole system was aerated and fresh NaOH (1 M) in a new vial was placed on the soil surface and covered with the plastic chamber. Similar measurement was carried out for the control treatment. Barium chloride (1 M BaCl2) was added to the solutions in the vials from the experimental plots to precipitate the carbonates to facilitate determination of CO2 evolved from the soil. The evolved CO2 was then determined by titration. Excess NaOH in solution was titrated against 1.0 M HCl using phenolphthalein indicator after precipitating the carbonate formed with 1.0 M BaCl2. Microbial metabolic quotient was thereafter calculated as the ratio of basal respiration to microbial biomass C (Anderson and Domsch 1990).

2.7 Cultivable bacterial and fungal biomass

Composite rhizospheric soil samples were collected at rice flowering stage and stored in a sterile container for cultivable fungal and bacterial analysis (Oladele and Ayodele 2017). Serial dilutions (up to 10−8) were prepared, and 100 μl from appropriate dilutions was dispensed on Nutrient agar for bacterial culture and Potato dextrose agar (PDA) for fungal culture. Cultivation for bacteria was done as described by Paul and Clark (1989). Inoculated plates were incubated at a temperature of 28 °C for 2 days and discrete colonies developed were counted. Cultivation and enumeration of fungi colonies was done as described by Harrigan and McCanne (1990). The inoculated plates were incubated at 28 °C for 7 days, and the total numbers of fungi were counted. For each dilution, respectively, number of colony on each individual plate was counted and amount of bacteria and fungi were calculated by using the following equation: CFU/g = (number of colonies × dilution factor)/volume of culture plate. Fungal-to-bacteria ratio was determined by direct microscopy and determined as the sum of total isolated fungal count divided by total bacteria and fungal count.

2.8 Extraction of native arbuscular mycorrhizae fungi (AMF) spores

AMF spores were isolated from rhizospheric soil samples collected at rice flowering stage by wet-sieving and decanting technique (Gerdemann and Nicolson 1963). Approximately 250 g of soil samples was suspended in 100 ml of water, and the heavier particles were allowed to settle for a few seconds. Suspended liquid was decanted to allow the desired spores to pass through. Spores were examined with a stereoscopic microscope (× 25 and × 50) with strong direct lighting. Density of spores in each soil sample was calculated by following Stahl and Christensen (1982) standard method.

2.9 Data analysis

Microbial biomass pools, viz (MBC, MBN and MBP), soil basal respiration, metabolic quotient, bacterial population, fungal population, fungal/bacteria ratio and native AMF spore count data were analysed by two-way factorial analysis of variance (ANOVA) to test for interactive and main effects of rice husk biochar and N fertilizer, and the means were compared with Tukey’s test at p < 0.05. A separate one-way ANOVA was conducted for the treatment combinations, where interactive effect was significant. Thereafter, significant differences amongst the treatment combinations were assessed using Tukey’s HSD test. Where necessary, data were log-transformed to reduce heteroscedasticity and improve assumptions of normality. All analyses were performed using the Statistical Analysis System package, version 9.2 (SAS Institute Inc 2008, SAS Institute Cary, NC). Means and standard errors were calculated to assemble graphs using Microsoft excel 2010.

3 Results

3.1 Microbial biomass carbon (MBC)

Biochar amendment, N fertilizer and their interactions significantly (p < 0.05) affected the soil MBC at 0–10 and 10–20 cm soil depth after 2 years of study (Table 1a, b). In the first year, significant (p < 0.05) effect of biochar × N fertilizer combination was recorded in MBC pool at the depth of 0–10 cm. Treatment combination N0B6 had the largest pool of MBC (Online resource 1). Main effect of biochar doses and N fertilizer rates showed inconsistent trends and were significant at 0–10 and 10–20 cm for biochar doses in the first year (Table 1a). At the depth of 10–20 cm, significant (p < 0.05) interaction of biochar × N fertilizer was observed (Online resource 2). In the second year of study, treatment combination N60B12 had the highest level of MBC which was 55% lower than the highest value recorded in 2016 at the soil depth of 0–10 cm (Online resource 3). Similar trend was observed at the soil depth of 10–20 cm as MBC pools were generally lower than that of the top 10 cm depth (Online resource 4). On the other hand, main effect of biochar doses and N fertilizer rates showed significant (p < 0.05) effects at 0–10 and 10–20 cm, respectively, (Table 1b).
Table 1

Effects of biochar, N fertilizer and interaction of biochar × N fertilizer on soil microbial biomass

Factors

0–10 cm

10–20 cm

Biochar rate (t ha−1)

MBC (µg g−1)

MBN (µg g−1)

MBP (µg g−1)

MBC/N ratio

MBC/P ratio

MBC (µg g−1)

MBN (µg g−1)

MBP (µg g−1)

MBC/N ratio

MBC/P ratio

a (2016)

0

325.6a

30.00a

1.82b

11a

178c

205.22a

21.36ab

1.56a

9a

131a

3

343.5b

28.60a

2.41d

12b

143a

252.10b

25.31b

1.72b

10a

143a

6

343.00b

38.28b

1.74a

9c

197d

301.53c

22.15ab

2.22c

13b

136a

12

335.5c

47.40c

2.06c

7d

163b

299.58c

19.50a

1.87b

15b

162b

N fertilizer (kg ha1)

0

338.5a

36.50b

1.93a

9a

175b

265.08a

20.47a

1.84ab

13a

143ab

30

338.8a

37.11b

2.10c

9a

165a

268.30a

23.49a

1.82a

11a

148b

60

332.7b

40.55c

2.03bc

10a

168ab

266.08a

22.23a

1.78a

12a

152b

90

337.6ab

30.15a

1.98ab

11b

174b

258.98a

22.13a

1.99b

12a

131a

P (F test)

Biochar

*

*

*

*

*

*

*

*

*

*

N rates

*

*

*

*

*

NS

NS

*

NS

*

B × N

*

*

*

*

*

*

NS

*

*

*

b (2017)

0

62.47b

7.76a

31.07a

9ab

2a

61.60b

15.33ab

0.67a

4a

162b

3

36.94a

5.95a

28.45a

7a

1a

32.10a

6.91a

0.92a

5a

88a

6

114.60c

12.90b

29.62a

10ab

3b

75.22c

17.46b

0.67a

5a

208c

12

139.10d

13.15b

26.17a

11b

6c

61.82b

12.34ab

0.70a

5a

170b

N fertilizer (kg ha−1)

0

90.26c

11.46a

24.05a

7a

4c

60.95b

11.32a

0.60a

5a

255c

30

65.80a

7.36a

30.07b

9a

2a

61.82b

14.95a

0.75a

5a

131b

60

79.00b

9.08a

29.90b

8a

2ab

45.92a

10.70a

0.80a

5a

84a

90

118.08d

11.85a

31.30b

11a

3bc

62.05b

15.08a

0.82a

5a

157b

P (F test)

Biochar

*

*

NS

*

*

*

*

NS

NS

*

N rates

*

NS

*

NS

*

*

NS

NS

NS

*

B × N

*

NS

NS

NS

*

*

*

NS

NS

*

Legend: NS = not significant

Means followed by the same letter are not significantly different from one another based on Tukey’s test at p ≤ 0.05

*Significant

3.2 Microbial biomass nitrogen (MBN)

Biochar amendment and N fertilizer interactions significantly (p < 0.05) affected the soil MBN at 0–10 cm in 2016 and 10–20 cm in 2017 only (Table 1a, b). In the first year, treatment combinations N60B12 accumulated the highest MBN pool at the top 10 cm soil depth (Online resource 1). Further, MBN pools were also found to be highest in treatment N30B3 at the depth of 10–20 cm though values recorded were lower than that of the top 10 cm depth of the soil (Online resource 4). Main effect of biochar doses and N fertilizer rates showed inconsistent trends and were significant at soil depths of 0–10 and 10–20 cm in biochar doses in the first year of study (Table 1a). In the second year, soil MBN pools were generally low in biochar and N fertilizer treatment combinations at the top 10 cm soil depth. This observation suggests rapid N mineralization and increased N availability for uptake by rice plant. However, treatment combinations N60B12 had the highest pool of MBN which was 92% lower than values recorded in 2016 (Online resource 3). At the depth of 10–20 cm, a slight accumulation of MBN was recorded as MBN pools were found to be highest in treatment combination—N0B6 (Online resource 4). Main effect of biochar significantly increased MBN with increasing doses at 0–10 cm, although an inconsistent trend was recorded at 10–20 cm soil depth (Table 1b).

3.3 Microbial biomass phosphorus (MBP)

Biochar amendment and N fertilizer interactions significantly (p < 0.05) affected the soil MBP at 0–10 and 10–20 cm only in the first year (Table 1a, b). Low levels of MBP pool were generally recorded amongst biochar and N fertilizer combinations at the soil depth of 10 cm (Online resource 1). However, at the soil depth of 10–20 cm, treatment combination N90B6 accumulated the highest pools of MBP (Online resource 2). Main effect of biochar doses and N fertilizer rates were both significant at the soil depth of 0–10 and 10–20 cm in the first year (Table 1a). However, expected corresponding increase in MBP pool with increasing doses of biochar and rates of N fertilizer were inconsistent. In the second year, MBP pool was slightly higher than values recorded in the first year of study at 0–10 cm soil depth (Online resource 3). On the other hand, main effect of N fertilizer rates was only significant at soil depth of 0–10 cm, while biochar amendment exerted no significant effect on MBP (Table 1b).

3.4 Microbial biomass C/N and C/P ratio

Biochar amendment and N fertilizer interactions significantly (p < 0.05) affected MBC/MBN ratio and MBC/MBP ratio at soil depths of 0–10 and 10–20 cm in the first year of study (Table 1a). In the second year, biochar amendment and N fertilizer interactions significantly (p < 0.05) affected the soil MBC/MBP ratio only (Table 1b). Variability in MBC/N ratio and MBC/P ratio was large amongst biochar and N fertilizer treatment combinations in both years of study (Online resource 1–4). In the first year, MBC/N ratio was found to be highest in treatment combination N60B3 at the top 10 cm soil depth (Online resource 1). Conversely, at the soil depth of 10–20 cm, higher values of MBC/N ratio were generally recorded when compared to values observed at the top 10 cm (Online resource 2). MBC/P ratio was found to be higher in treatment combinations N30B6 in the first year of study at 0–10 cm depth, while N60B12 had the largest MBC/P ratio at the soil depth of 10–20 cm (Online resource 1, 2). Follow-up studies in the second year showed similar large variability amongst treatments as observed in the first year. However, ratios recorded were generally lower than MBC/N ratios in the first year (Online resource 3). Surprisingly, MBC/N ratio was also increased at the 10–20 cm depth of the soil (Online resource 4). Similar trends were observed with MBC/P ratios as values recorded were generally higher at 10–20 cm depth, while a reverse trend was observed at 0–10 cm soil depth (Online resource 1–4).

3.5 Soil respiration

Biochar amendment and N fertilizer interactions significantly (p < 0.05) affected soil CO2 flux at 0–10 and 10–20 cm soil depth after 2 years of study (Figs. 1, 2). Soil CO2 flux was observed to have varied significantly (p < 0.05) amongst treatment combinations. At the rice post-seeding stage (30 days after seed germination), CO2 flux was maximum amongst all treatment combinations. However, treatments in which N fertilizer was applied singly at the rate of 60 kg ha−1 without biochar combination (N60B0) evolved the largest CO2 flux at the soil depth of 0–10 cm. At the active tillering and harvest stage of rice plant, CO2 flux showed declining trends which suggest a decrease in the rate of CO2 emission (Fig. 1). In the subsurface layer (10–20 cm), CO2 flux was surprising increased especially in N fertilizer treatments applied singly (Fig. 2). At the post-seeding stage of rice plant growth, CO2 emission increased in treatments with singly applied N fertilizer in the order of—N0B0 > N30B0 > N60B0 > N90B0 (Fig. 2). On the other hand, treatments with combination of biochar and N fertilizer irrespective of doses and rates showed decreasing trend of CO2 flux (Fig. 2). At the active tillering and harvest stage of rice plant, CO2 flux was generally reduced in all treatments which suggest decreasing rates of microbial respiratory activity at both stages of rice plant cycle. However, we observed an increase in CO2 flux from treatment N0B3 at this stage of rice growth (Fig. 2).
Fig. 1

Effect of biochar × N fertilizer on soil CO2 efflux (0–10 cm). Error bars indicate 95% confidence interval for means as tested by two-way ANOVA (p < 0.05). (2017) Legend: N0 = 0 N kg ha−1, N30 = 30 N kg ha−1, N60 = 60 N kg ha−1, N90 = 90 N kg ha−1

Fig. 2

Effect of biochar × N fertilizer on Soil CO2 efflux (10–20 cm). Error bars indicate 95% confidence interval for means as tested by two-way ANOVA (p < 0.05). (2017) Legend: N0 = 0 N kg ha−1, N30 = 30 N kg ha−1, N60 = 60 N kg ha−1, N90 = 90 N kg ha−1

3.6 Soil microbial metabolic quotient (qCO2)

Two-way analysis of variance showed significant (p < 0.05) effect of treatment combinations on qCO2. Biochar amendment and N fertilizer interactions significantly (p < 0.05) affected the soil qCO2 at rice harvest stage in the top10 cm depth after 2 years of study (Fig. 3). Generally, at the surface layer of the soil, low level of qCO2 was recorded in all treatment combinations. However, treatments containing N fertilizer alone had slightly higher levels of qCO2. Our result showed increasing trend of qCO2 with increasing rates of N fertilizer applied without biochar combination (Fig. 3). Treatment combination N90B0 recorded the highest level of qCO2, while biochar and N fertilizer treatment interactions were generally lower irrespective of doses or rates.
Fig. 3

Effect of biochar × N fertilizer on metabolic quotient. Different letters above columns indicate significant differences between treatments as tested by two-way ANOVA (p < 0.05). Error bars indicate 95% confidence interval for means (2017). Legend: N0 = 0 N kg ha−1, N30 = 30 N kg ha−1, N60 = 60 N kg ha−1, N90 = 90 N kg ha−1

3.7 Cultivable bacterial and fungal population

Biochar amendment, N fertilizer and their interactions significantly (p < 0.05) affected cultivable bacterial and fungal population in the first year, while fungal population and AMF spore count were significantly (p < 0.05) affected in the second year (Table 2). In the first year of study, we observed significant (p < 0.05) interactive effect of biochar and N fertilizer on fungal, bacterial population and fungal/biochar ratio (Online resource 5, Fig. 4). Contrasting results were recorded in the second year of study as treatment combinations exerted significant (p < 0.05) effect on fungal population, AMF spore count and fungal/bacteria ratio alone. Fungal population was markedly increased by 74% in the second year, while AMF spores was also increased and recorded the highest count in treatment N30B3 (Online resource 5). Startlingly, our result showed a shift in microbial community structure as we recorded a significant 96% increase in Fungi/bacteria ratio in the second year (Fig. 4).
Table 2

Effects of biochar, N fertilizer and interaction of biochar × N fertilizer on culturable bacterial, fungal (× 10−8) and AMF spore count

Factors

2016

2017

Biochar rate (t ha−1)

Bacteria (cfu g−1 soil)

Fungi (cfu g−1 soil)

AMF (spore 50 g)

Bacteria (cfu g−1 soil)

Fungi (cfu g−1 soil)

AMF (spore 50 g)

0

5.00a

3.20b

40.00a

4.83a

10.61a

23.00a

3

5.36a

1.92a

42.50a

4.96a

13.36a

64.25c

6

5.83a

2.37a

40.25a

4.76a

10.96a

57.75ab

12

5.13a

1.98a

44.00a

5.51a

11.04a

46.25b

N fertilizer (kg ha1)

0

4.76a

1.61a

43.25a

5.46a

9.88a

37.00a

30

5.40ab

3.09c

43.00a

4.67a

11.93a

43.75ab

60

5.40ab

2.14b

40.75a

4.58a

11.21a

50.50ab

90

5.75a

2.63b

39.75a

5.36a

12.95a

60.00b

P (F test)

Biochar

NS

*

NS

NS

NS

*

N rates

*

*

NS

NS

NS

*

B × N

*

*

NS

NS

*

*

Means followed by the same letter are not significantly different from one another based on Tukey’s test at p ≤ 0.05

Means followed by the same letter are not significantly different from one another based on Tukey’s test at p ≤ 0.05

*Significant

Fig. 4

Effect of biochar × N fertilizer on fungal/bacterial ratio. Different letters above columns indicate significant differences between treatments as tested by two-way ANOVA (p < 0.05). Error bars indicate 95% confidence interval for means. Legend: N0 = 0 N kg ha−1, N30 = 30 N kg ha−1, N60 = 60 N kg ha−1, N90 = 90 N kg ha−1

4 Discussion

4.1 Microbial biomass pools

Soil microbial biomass is an important indicator used widely for monitoring rapid changes in soil quality resulting from various anthropogenic influence and land management practices (Suman et al. 2006). MBC, MBN and MBP constitute about 3–7% of soil organic carbon (Corg), 1–5% of total soil nitrogen (TN) and 1–5% of total soil phosphorus (TP), respectively (Wu 2007). Changes in soil microbial biomass pools reflect the process of microbial growth, mortality and carbon mineralization (Zhang et al. 2014). Findings from our study clearly demonstrate that combination of biochar and N fertilizer resulted in increased pools of MBC and MBN especially at the top soil (0–10 cm) layer. However, low pools of MBP was observed after 2 years of study which suggests a high rate of P turnover possibly from organic P fraction of SOM, solubilization of mineral bound P, less P fixation and immobilization as evidently shown in the high level of measured available Bray-1 P (Online Resource 1–4). Increase in microbial biomass pools as influenced by biochar and N fertilizer combination has implications for microbial communities associated with rain-fed rice, which will in turn affect nutrient cycling, GHGs emission and Corg mineralization. Our study has shown that biochar incorporated into soils can serve as a porous N sorption medium for N fertilizer and other nutrient sources, thereby creating a nutrient dense micro-environment that is ideal for microbial growth. This could possibly explain the initial increase in MBC and MBN pools after biochar and N fertilizer application. Dempster et al. (2011) reported that MBC significantly decreased with biochar addition, while MBN was unaltered in a coarse-textured soil. In contrast to these earlier findings, Lehmann et al. (2011), Kolb et al. (2009), O’Neill et al. (2009) and Liang et al. (2010) corroborated our study by reporting similar positive effects of biochar addition on microbial biomass pools. Steiner et al. (2008) in a study reported a positive linear relationship between microbial biomass and biochar concentration in a degraded soil. Furthermore, Zhang et al. (2014) reported that 4 years of consecutive wheat straw biochar application increased MBC at 0–30 cm depth of the soil when applied at high dosage (9.0 t ha−1), but decreased MBC when low dosage (4.5 t ha−1) was applied. This is in contrast to our study, where MBC and MBN was significantly increased in the top 10 cm and reduced at the 10–20 cm soil depth (Online resource 1, 2). Although MBC was slightly decreased in the second year of study, we attribute this to sorption of labile/soluble organic carbon into protective pore spaces of biochar in amended soils. This protected organic carbon will cause a reduction in substrate availability required by soil microbes for energy generation and respiratory activity. A similar trend was observed in MBN pools which suggest high N turnover rate from organic N mineralization. The increase in MBC and decrease in MBN in this study indicate that combination of biochar and N fertilizer acted more as a carbon sink rather than nitrogen source for soil microbes with consequent implication for N availability (Zhang et al. 2014).

4.2 Soil microbial biomass C/N and C/P ratios

MBC/N and MBC/P ratios are important indices for determination of availability of carbon and nitrogen to soil microbes and changes in biomass composition. Large ratios of MBC/N and MBC/P (resulting from biochar- high carbon content and N fertilizer- soluble N) in the first year contributed substantially to N and P immobilization and retention as evidently shown by the high level of soil total nitrogen and available phosphorus (Online resource 1–4). Increased MBC/N ratio suggest N immobilization, although it is uncertain if this increase was due to changes in microbial community structure, soil properties or environmental factors in amended treatments. However, in the second year at the soil depth of 10–20 cm, MBC/N and MBC/P ratios decreased due to lower pools of MBC, MBN and MBP which induced an increase in flux of mineral N and P (Online resource 1–4). This has implications for C and N biogeochemical cycles in humid tropical alfisol under rain-fed rice cultivation in the context of C sequestration and N mineralization. Furthermore, we argue that a stimulatory effect of biochar and N fertilizer addition on recalcitrant C, organic N and P pools could have induced reduction in MBC/N and MBC/P ratios in the second year of study. The mechanism behind these reduced microbial ratios can be attributed to combination of N fertilizer with biochar which stimulated available N within the pore sites of biochar in amended treatments. Similar study corroborating our findings was reported by Li et al. (2016) who found an increase in MBC/N, MBC/P and MBN/P ratios in a long-term experiment under combinations of organic and inorganic soil amendment in a double rice cropping system in China. They went on to further suggest the use of MBC/N and MBC/P ratios as an indicator of soil productivity for rice fields. Conversely, Kushwaha et al. (2000) reported a decrease in MBC/N ratio after incorporation of rice straw residues, while Kallenbach and Grandy (2011) reported no effect on MBC/N ratio after application of carbon dense organic amendment, suggesting that changes in MBC/N ratio cannot be attributed to amendment of soils with organic biomass containing relatively high C content only.

4.3 Soil respiration

Soil CO2 flux are useful indicators of microbial activity and changes occurring in the soil due to the addition of organic and inorganic amendment, land use and management (Jin 2010). These changes are of vital importance in determining carbon sequestered or evolved in the context of climate change and soil quality improvement in the tropics. Soil CO2 flux is generally modulated by plant roots, soil microbes, soil properties, plant growth stages and their interactions (Epron et al. 2004). Thus, plant growth stages may have an important role in modulating soil CO2 efflux through its influence on rhizospheric activities (Shi et al. 2006). Soil CO2 flux was observed to increased initially during rice post-seeding, tiller initiation and stem elongation stages due to improved soil moisture conditions and vigorous root development (Figs. 1, 2). However, a slight decrease in CO2 flux was recorded during active tillering stage which suggests decreasing rhizospheric activity and more nutrient transfer to plant for increased growth and development. At harvest stage, further reduction in soil CO2 flux measured could be attributed to decreased release of plant root exudates and labile C from rice plants for soil microbes due to the plant being at its latter stage of phenological cycle. Furthermore, we argue that plausible decrease in soil moisture at this stage of rice plant could have contributed to reduced microbial activities. Soil water content and temperature are regarded as major drivers of CO2 flux as it is observed that an increase in soil moisture with optimum soil temperature range can influence soil microbial activities thereby resulting in high soil CO2 flux (Srivastava et al. 2017). Treatments with N fertilizer only at rates between 60–90 kg ha−1 and their combination with biochar dose of 3 t ha−1 had the highest soil CO2 flux at post-seeding stage of rice plant at 0–20 cm depth. This suggests some sort of positive priming effect of N fertilizer on native Corg with implications for carbon sequestration. However, it is pertinent to note that this effect could be temporary or transient. Our finding is in contrast to studies conducted by Agegnehu et al. (2016) and Srivastava et al. (2016a) who reported reduced soil CO2 flux from mineral fertilizer treatments when compared with farm yard manure and rice husk ash in a tropical aridisol. In addition, a study by Smith et al. (2010) reported higher CO2 flux initially in biochar-amended soils which declined after 1 week of application. Usually, high CO2 flux is evolved in biochar-amended soils which may be increased or decreased depending on the availability of labile carbon content of incorporated biochar (Jones et al. 2012; Mohan et al. 2018). The initial increase in CO2 suggest higher soil microbial activities and nutrient cycling which may get stabilized leading to storing of Corg in soil aggregate fractions (Srivastava et al. 2016b).

4.4 Microbial metabolic quotient (qCO2)

Our study monitored the dynamics of soil basal respiration and its relationship with microbial biomass pools to provide more understanding into changes in microbial activity associated with soils amended with biochar and N fertilizer combinations. The mechanism behind decreasing soil qCO2 as biochar doses increased irrespective of N fertilizer addition could be that the incorporation of biochar containing a stable and recalcitrant fraction of carbon, and large C/N ratio altered the cycling of carbon dense compounds mediated by microbial processes. Our observation was consistent with the findings of a field study conducted on a charcoal laden anthrosol in the Central Amazon, Brazil, where the anthrosols were observed to have about 80% lower CO2 evolution per unit carbon over 1 year and 6 months compared to their respective adjacent soils with low charcoal contents (Liang et al. 2008; Jin 2010). The lowest qCO2 values were measured in soils that received biochar and N fertilizer combination irrespective of doses and rates. The increased MBC, but decreased microbial respiration observed in biochar-amended treatments in our study suggests that the microbes in the amended soils produced more cell mass per unit of carbon degraded than un-amended treatment. This imply that soil microbes degraded less carbon and tended to adsorb carbon as evidently shown by increase in microbial biomass in response to biochar addition. In other words, biochar amendment enhanced microbial carbon use efficiency and increased Corg retention in soil (Jin 2010). It also indicated that biochar-amended soils may play a better role in protecting microbes from disturbance or stress. Lower qCO2 may also indicate low activities of microbes in biochar-amended soils compared to non-amended soils or a shift of Corg towards recalcitrant fraction difficult for microbial degradation (Six et al. 2006).

4.5 Cultivable bacterial and fungal biomass

Our finding clearly shows that combination of biochar and N fertilizer increased cultivable rhizospheric bacterial population associated with rain-fed rice in the first year of study (Online resource 5). We attribute this to favourable niche conditions created by this amendment combination such as increased soil pH, low soil C/N ratio, readily available nutrients and labile substrate sources of food and energy required for microbial activity (Allison et al. 2005). This significant increase observed in population of cultivable bacteria suggests a possible suppression of fungal population due to probable antagonistic interactions of microbes (Egamberdieva et al. 2016). However, this increase in bacteria population could create some ecosystem problems with implications on nutrient cycling and rapid decomposition of native Corg consequently offsetting carbon sequestration. These findings are in agreement with Chen et al. (2013) who reported an increase in bacterial abundance in an acidic rice paddy field amended with biochar in south-west China. Furthermore, Kolton et al. (2011) and Egamberdieva et al. (2016) reported similar findings by observing a change in total root-associated microbial community composition of sweet pepper and soybean after amendment with biochar from citrus wood and maize cob feedstock and hydrochar. However, in the second year of study, we observed a shift to a more fungal-dominated population (Online resource 5, Fig. 4). We attribute this change to the depletion of labile substrates such as carbon which is important for bacterial community’s growth and respiratory activity. We also observed an increase in fungal population in singly applied N fertilizer treatments. This is surprising and requires further verification to elucidate possible mechanisms. However, we argue that an increase in fungal population in N fertilizer treatments applied at lower rates (N30B0) could be the result of negative priming effect on native Corg content which induced an increase in fungal population. This is because fungi are widely known to have higher carbon assimilation efficiency (Lundquist et al. 1999). This submission was also corroborated by the study of Nakhro and Dkhar (2010) who reported that increasing fungal population showed positive correlation with Corg in organic and inorganic fertilized paddy field in India. Combinations of biochar and N fertilizer induced an increase in fungal/bacteria ratio which could be associated with the ash-rich content of rice husk biochar and a corresponding increase in total Corg availability. Furthermore, increase in the ratio of fungal to bacteria could also be attributed to the greater growth efficiency of fungi and the accumulation of carbon in the less decomposable fungal biomass. Fungi, with their far-reaching hyphaes, can form thread like attachments on biochar creating bridges between microbes and host plants, allowing the use of C exudates from plants instead of C decomposition (Jin 2010). The greater growth efficiency of fungi and the accumulation of carbon in their biomass for cell growth give fungi greater carbon use efficiency over bacteria. Biochar upon incorporation in the soil sorbs nutrients, enzymes and create liveable and favourable habitat for fungi proliferation relative to bacterial growth. The rice husk biochar applied in this study had a pH of 8.50, and the alkaline conditions resulting from biochar amendment may favour fungal growth over bacterial growth as fungi’s are more tolerant of the alkaline environment created by biochar than bacteria. Increasing fungi/bacteria ratios as observed in our study could indicate a robust and resilient soil ecosystem with important benefits provided such as abiotic and biotic stress tolerance, efficient water and nutrient uptake by plants modulated by some select fungal taxa (Bougnom et al. 2010).

4.6 Native AMF spore count

We argue that high level of available P in biochar-amended plots could have inhibited AMF sporulation and colonization activity in the first year of study (Online resource 5). Phosphorus availability is central to AMF sporulation and association with plants (Smith and Read 2008). Studies have suggested that either extremely low (e.g. Allen et al. 2003; Drew et al. 2006) or high (Corbin et al. 2003; Covacevich et al. 2006; Gryndler et al. 2006) soil available P can adversely affect AMF abundance in roots and soils. In contrast to what was reported in previous studies, biochar and N fertilizer combinations had no effect on native AMF spore abundance in the first year. This could be attributed to the ability of freshly incorporated biochar to display neutral or detrimental effect on the activity of other soil microbes with direct and indirect effects on AMF germination. For instance, mycorrhization helper bacteria (MHB) or phosphate solubilizing bacteria (PBS) are capable, under certain conditions, of secreting metabolites such as flavonoids, that stimulate the multiplication of AMF spores and the subsequent colonization of plant roots (Warnock et al. 2007). This is further corroborated by Hildebrandt et al. (2006) who observed that certain compounds such as raffinose and other metabolites secreted by select strains of Paenibacillus can directly enhance the growth of AMF spores and extraradical mycelium. Though a dominant cultivable bacterial population in the first year of study was recorded, we suspect that the dominant taxa of the bacterial communities were not MHBs or PBS as evidently supported by low AMF spore population. Furthermore, biochar sorptive capacity in adsorbing signalling compounds released from rhizospheric zone of rice plant roots which stimulates sporulation could decrease the ability of AMF to colonize plant roots. The presence of soluble phenols in slowly pyrolysed biochar produced under low temperature as such used in our study may have been toxic to native AMF communities thereby inhibiting initial sporulation. This argument was corroborated by Wallstedt et al. (2002) who reported that the addition of activated charcoal slurry to an experimental soil resulted in decreased amount of water-soluble phenols which stimulated increased AMF sporulation and ectomycorrhizal fungus colonization of plant roots. Surprisingly, in the second year of study, AMF spore count increased despite high soil available P. The reason for this is still unclear; however, studies by Matsubara et al. (2002) and Yamato et al. (2006) have shown that increased pH level after biochar amendment to soil might be responsible for influencing AMF abundance. It is important to note that this study was short term and the response of microbial communities to changes in soils amended with biochar and availability of nutrient as biochar ages remains elusive.

5 Conclusions

After 2 years of study, our results indicate that combination of rice husk biochar and N fertilizer caused an initial significant increase in soil MBC, MBN and MBP pools, while a slight decrease was observed in the second year. We also showed that increased MBC/N and MBC/P ratio in the first year of study and a corresponding decrease in the second year suggests that large ratios of MBC/N and MBC/P resulting from biochar- high carbon content and N fertilizer- soluble N contributed substantially to N and P retention as evidently shown by the high level of soil total nitrogen and available phosphorus. Furthermore, combination of biochar and N fertilizer stimulated microbial activity by increasing microbial biomass and respiratory activities (CO2 flux) predominantly at the post-seeding stage of rice plant. However, soil microbial metabolic quotient (qCO2) which monitors respiration rate per unit of microbial biomass or a reflection of microbial efficiency was greatly reduced. This suggests enhanced carbon utilization by soil microbes and bodes well for the drive towards carbon sequestration in tropical soils. Combinations of biochar and N fertilizer induced a shift to a more fungal-dominated population during the 2 year study. This shift in community composition is expected to help aid in retention of soil organic carbon as fungi have higher carbon assimilation efficiencies than bacteria. Future studies should focus on long-term implications and microbial community structure stability of soils amended with biochar and nitrogen fertilizer.

Notes

Compliance with ethical standards

Competing interests

The authors have declared that no competing interests exist.

Supplementary material

42773_2019_17_MOESM1_ESM.docx (38 kb)
Supplementary material 1 (DOCX 37 kb)

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Copyright information

© Shenyang Agricultural University 2019

Authors and Affiliations

  • Segun Oladele
    • 1
    • 2
    Email author
  • Adebayo Adeyemo
    • 2
  • Ajoke Adegaiye
    • 2
  • Moses Awodun
    • 2
  1. 1.Department of AgronomyAdekunle Ajasin University Akungba AkokoOndo StateNigeria
  2. 2.Department of Crop, Soil and Pest ManagementFederal University of Technology AkureOndo StateNigeria

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