Volume 10, Number 2 (January 2023):4271-4280, doi:10.15243/jdmlm.2023.102.4271 ISSN: 2339-076X (p); 2502-2458 (e), www.jdmlm.ub.ac.id
Open Access 4271 Research Article
Reducing Pb accumulation in roots of sweet potato under low lead-contaminated soil by Azotobacter inoculation
Reginawanti Hindersah1*, Vera Oktavia Subarja2, Pujawati Suryatmana1, Rija Sudirja1, Agung Karuniawan3, Yusup Hidayat4
1 Department of Soil Science, Faculty of Agriculture, Universitas Padjadjaran, Jl. Raya Jatinangor Km 21 Jatinangor 40600 Bandung, Indonesia
2 Department of Soil Science, Faculty of Agriculture, Karawang University, Jl. HS. Ronggo Waluyo, Puseurjaya, Telukjambe Timur, Karawang 41361, Indonesia
3 Department of Agronomy, Faculty of Agriculture, Universitas Padjadjaran, Jl. Raya Jatinangor Km 21 Jatinangor 40600 Bandung, Indonesia
4 Department of Plant Pest and Diseases, Faculty of Agriculture, Universitas Padjadjaran, Jl. Raya Jatinangor Km 21 Jatinangor 40600 Bandung, Indonesia
*corresponding author: reginawanti@unpad.ac.id
Abstract Article history:
Received 24 September 2022 Accepted 21 November 2022 Published 1 January 2023
Agricultural soil is possibly threatened by lead (Pb) contamination due to the intensive use of fertilizers. The rhizobacteria were recommended for the bioremediation of soils contaminated by low concentrations of Pb. The experiment was conducted to observe the Azotobacter's ability to proliferate in Pb-contaminated broth and to decrease the Pb availability in soil, Pb uptake by sweet potato roots, and sweet potato growth. The resistance test was performed by growing five Azotobacter isolates in N-free broth with various Pb levels. A pot experiment was conducted in a factorial randomized block design to test three levels of Pb in soil and two Azotobacter isolates. The results showed that Azotobacter Azv4 and A. choroococcum were resistant to 100 mg L-1 Pb in N-free broth. In the pot experiment, Azotobacter Azv4 Inoculation caused less Pb in soil and roots of sweet potatoes grown in Pb-contaminated soil than A. choroococcum.
Either Azotobacter or Pb soil did not influence vine length. However, Azv4 was more prominent in increasing branch number, root volume and length;
higher Pb in soil reduced branch number but did not affect root parameters.
Azotobacter Azv4 increased more shoot and root dry weight compared to A. choroococcum, but both isolates did not change the shoot-to-root ratio (S/R). The Pb contamination only reduced root dry weight and reduced the S/R. This research considered utilizing rhizobacteria Azotobacter for reducing Pb levels in soil and roots; and increasing sweet potato biomass.
Keywords:
Azotobacter biomass Pb resistance Pb uptake soil Pb
To cite this article: Hindersah, R., Subarja, V.O., Suryatmana, P., Sudirja, R., Karuniawan, A. and Hidayat, Y. 2023. Reducing Pb accumulation in roots of sweet potato under low lead-contaminated soil by Azotobacter inoculation. Journal of Degraded and Mining Lands Management 10(2):4271-4280, doi:10.15243/jdmlm.2023.102.4271.
Introduction
Lead (Pb) is possibly a toxic heavy metal since the metal has no essential function to an organism, even at very low concentrations. Plants do not require Pb for their metabolism; therefore, certain concentrations of Pb potentially threaten plant development growth.
Many agricultural areas in Java Island, Indonesia dominated by volcanic soils. The Pb and their isotopes are naturally found in volcanic parent materials in Java (Handleym et al., 2014).
Increased Pb level in soils is evident elsewhere due to anthropogenic activities, including food crop cultivation. Lead contamination in soil may be caused
Open Access 4272 by the use of fertilizers, mainly rock phosphate-based
fertilizers. The Pb impurities are found in superphosphate, simple phosphate, triple superphosphate, potassium phosphate and even in Urea and NPK fertilizer (Benson, 2014, Azzi et al., 2017; Kratz et al., 2017). The intensive and high amount of N and P fertilization to induce Pb accumulation in biomass have been reported in the subtropical monsoon climate of Pakistan and China (Al-Hadi et al., 2014; Wei et al., 2020) as well as in subtropical desert Iraq (Al-Qasi et al., 2021).
Increased Pb content in agricultural areas poses a serious problem not only for sustaining food crop production but also for human health. Once growing plant in Pb-contaminated soil, plant roots uptake the ionic Pb through membrane transporters mechanism by different plant proteins (Williams et al., 2000; Jain et al., 2018). Biomagnification of toxic heavy metals in plant tissues leads to the reactive oxygen species accumulation that induces the reaction of the ageing process (Jalmi et al., 2018). The phytotoxicity of high concentration of Pb in soil resulted in a decrease in plant growth and productivity due to the disruption of cell membrane permeability and the decline in photosynthetic rate (Hadi and Aziz, 2015; Nas and Ali, 2018).
Food crop cultivation without NPK fertilizers might preclude Pb accumulation in soil, but in the tropics, it potentially reduces yield. Low nitrogen efficiency due to the leaching process and phosphorus adsorption in tropical sail resulted in low N and P in soil (Almaz et al., 2017; Hanyabui et al., 2020).
Therefore, the application of a more environmental- friendly N source to replace chemicals is needed.
Nowadays, biofertilizers have become an important part of nutrition management in sustainable agriculture. Certain strains of free-living N2-fixing bacteria are considered to include in biofertilizer formulation.
The Plant growth-promoting rhizobacteria (PGPR) has been recommended for low-cost bioremediation of HM-contaminated soils.
Azotobacter is Gram Negative bacteria with pleomorphic cell morphology and forms cysts under drought environments (Upadhyay et al., 2015;
Mukhtar et al., 2018). The genus of N2-fixing Azotobacter is well-known PGPR that enhances plant growth through N2 fixation, phosphorus solubilizing as well as phytohormone, and exopolysaccharide (EPS) production (Hindersah et al., 2020). The natural characteristic of Azotobacter is capsule formation consisting of EPS considered as an essential substance in metal resistance mechanisms (Rasulov et al., 2013;
Dhevagi et al., 2021). In relation to Pb availability for plant uptake, an increase in EPS produced by Azotobacter causes more cationic Pb bound by EPS, and they become immobile and unavailable for plants (Dhevagi et al., 2021).
Azotobacter is now broadly used as PGPR in tropical crop cultivation. Many researchers have
demonstrated the ability of Azotobacter to improve tuberous plant growth, biomass and production (Hussien and Ghazi, 2016; Abedaboohanah et al., 2020; Hindersah et al., 2021). Increased root biomass after Azotobacter inoculation due to phytohormone production has been documented (Van Oosten et al., 2018). Better plant rooting resulted in the increment of root hair for nutrient uptake and also root exudation that provides more nutrients for microbial proliferation in the rhizosphere (Holz et al., 2017; Upadhyay et al., 2022). Therefore, Azotobacter benefit plant grown in heavy metal-contaminated soil not only by reducing heavy metal availability but also increase plant growth.
Lead transfer from soil to plant biomass is highly dependent on soil reaction. Lead availability in soil is enhanced by lower pH and N fertilizer (Shi et al., 2013;
Wierzbowska et al., 2018; Wei et al., 2020). The bioavailability of Pb in acidic soil is higher and enhances exchangeable Pb (Yan et al., 2017). The roots, tubers, stems and leaves accumulated Pb in significant concentration when the soil contains a high amount of Pb (Keran et al., 2008; Kuntal Shah et al., 2017). The average level of Pb detected in the commercialized sweet potato was 0.003 mg kg-1, below the toxicological risk to consumers (Luis et al., 2014). However, some studies reported the high Pb content of sweet potatoes cultivated in a certain agroecosystem (Alias, 2013; Pembere, 2018).
Sweet potatoes are produced in almost all regions of Indonesia. Even though it is not the main carbohydrate source in the diet, tubers are prominent in the food industry. The Pb content in roots should be considered to determine the potency of Pb accumulation in the tuber since Pb uptake by roots is the only way to transfer soil Pb to the tubers and shoots. Despite the importance of sweet potato for both foodstuff and industry in Indonesia, the study related to growth and the Pb uptake in Pb-contaminated soil is rare. In an agricultural area where the level of metal contamination is usually not high, bioremediation is considered an effective way to clean the soil. The objectives of this pot experiment were 1) to analyze the cell viability of Azotobacter isolates grown in liquid media contaminated with several concentrations of Lead nitrate; and 2) to verify the ability of Azotobacter spp. to reduce the lead availability in soil and its uptake by roots, and their effect on sweet potato growth during the vegetative stage. Although the Pb content, in general, is lower than the threshold concentration, this result of the experiment provides a basis for preventing the Pb content increment in tuberous food crops with intensive fertilization.
Material and Methods
The research was conducted on November 2020 to January 2021. The laboratory experiment was done at the Soil Laboratory of Universitas Karawang, West Java. The pot trial was conducted in the field experiment at the Faculty of Agriculture, Jatinangor
Open Access 4273 Campus, Universitas Padjadjaran, West Java. The area
is located in the tropics with an altitude of 725 m above sea level. The minimum and maximum temperature and relative humidity during the experiment are 14.3 oC and 27.8 oC; and 77.6% and 96.3%, respectively. The rainfall during the three months is 358 mm. The climate data were provided by Meteorological Station in Jatinangor.
Resistance test on Azotobacter isolates
The Azotobacter Azv1 and Azv2 were isolated from paddy rhizosphere, Azv3 and Azv5 were obtained from hydrocarbon-contaminated soil, while Azv4 was isolated from saline soil. The A. chroococcum (AC) isolated from corn rhizosphere was provided by Universitas Padjadjaran. The pure cultures of all strains were transferred to the N-free Ashby’s slant for
three days at 30 oC before being used in the Pb-resistance test. The composition of media was 20 g
Mannitol, 0.2 g K2HPO4, 0.2 g MgSO4, 0.2 g NaCl, 0.1 g K2SO4, and 5 g CaCO3.
The resistance test on Pb(NO3)2 was performed by growing six Azotobacter strains in N-free Ashby broth contaminated with various concentrations of Pb. In the present assay, the 100 mL broth in a 250-mL Erlenmeyer flask was added with Pb(NO3)2 in different concentrations, i.e. 1.0, 10 and 100 mg L-1 Pb and inoculated with 5% Azotobacter liquid culture with a density of 3.2 x 107 CFU mL-1. The control treatment was without Pb contamination. All treatments were replicated three times. The cultures were incubated for 7 days at room temperature (24 oC ± 2) on a 115-rpm gyratory shaker (Gerhardt RO-2). At the end of incubation, the Azotobacter population was counted by serial dilution plate method on Ashby’s medium.
The data of each Azotobacter population was subjected to mean and standard deviation calculation and presented in Table 1.
Pot experiment
The soil used in the pot trial was silty clay loam Inceptisols with pH of 6.40, low organic-carbon (1.66%), average total-Nitrogen (0.24%), low C to N ratio (7), low available P2O5 (5.75 mg kg-1), average potential P2O5(25.75 mg 100 g-1), and low potential K2O (14.49 mg 100 g-1). The cation exchange capacity (CEC) and base saturation (BS) of soil were high (38.04 cmol kg-1) and average (44.74%), respectively
(PPT, 1983). The soil naturally contained Pb 17.7 mg kg-1, lower than the minimum threshold of
Pb in soil (100 mg kg-1) recommended by the Indonesian Ministry of Environment. The chicken manure had a pH of 6.7 and contained 14.41% organic C, 1.25 % total N 1.25, C/N ratio of 11.52, 1.69% total P, 1.21% total K, C/N of 11.52 and 28.1% water content. The Pb content of manure was not detected based on Atomic Absorption Spectroscopy analysis.
Based on the previous laboratory experiment, Azotobacter sp. Azv4 isolated from saline soil and A. chroococcum were the most resistant to
100 mg kg-1 Pb. Both Azotobacter (Figure 1) were used in the pot experiment. The Azv4 isolates grown in N-free Ashby broth produced 2.64 mg L-1 Indole Acetic Acis (IAA), 1.0 mg L-1 Gibberelins (GAs), 0.37 mg L-1 Zeatin, 0.72 mg L-1 kinetin, and 5.1 g L-1 EPS; and has nitrogenase activity of 7 nmol acetylene g-1 h-1. In the same broth, A. chroococcum excreted 0.52 mg L-1 IAA, 0.41 mg L-1 GAs, 0.97 mg L-1 CKs and 6.4 g L-1 EPS with nitrogenase activity of 74.1 nmol ethylene g-1 h-1.
Experimental establishment
The pot experiment was set up in a completely randomized block design to test two factors. The first factor was NFB inoculation consisting of control (without N-fixing bacteria), Azotobacter sp. Azv4 and A. chroococcum. The second factor was Pb spiked soil
in the pot consisting of 27.04 mg kg-1 and 37.02 mg kg-1 Pb. All the combination treatments were
replicated three times. The soil was collected from 30-cm depth topsoil, cleared up from plant debris and roots, and air-dried for two days. A total of 9.5 kg of soil were mixed with 0.5 kg of chicken manure and then put in 30 cm x 30 cm perforated black polyethylene bag. Chicken manure was added since the soil has low organic carbon, and the manure provides organic carbon for Azotobacter. All bags were placed in the open field without shade since the experiment was conducted in the dry season and during the pandemic C-19.
Azotobacter inoculation
Each Azotobacter isolate was grown separately in Ashby’s broth with the initial concentration of 1%
(v v-1) for three days at room temperature (24 0C ± 2) on a gyratory shaker (Gerhardt RO-2) of 115 rpm.
A total of 1% liquid inoculant was then transferred to sterilized 2% molasses broth enriched with 0.5%
NH4Cl and incubated for another three days at room temperature. The final Azotobacter population in molasses-based liquid inoculant was 107 colonies forming unit (CFU) mL-1. Azotobacter was applied by pouring 20 mL inoculant evenly on the surface of potted soil two days before planting. Control pots received a similar volume of sterilized distilled water.
Lead contamination
The spiked soil was mixed with 125 mg and 259 mg PbCl2, which contained 74.46% of Pb. The PbCl2 was then dissolved in 1,000 mL of sterilized distilled water.
The solution was poured into each pot containing 10 kg growth media, so the Pb content in soil from PbCl2 was 9.3 mg kg-1 and 19.28 mg kg-1, respectively.
The Pb in the soil before the experiment was 17.74;
then, after Pb spiking, the final Pb content of Pb spiked soil was 27.04 mg kg-1 and 37.02 mg kg-1,, which is
under the minimum threshold of Pb in soil (100 mg kg-1) according to the Ministry of State for
Population and Environmental of Indonesia, and Dalhousie University Canada (1992). The diluted
Open Access 4274 Pb(Cl)2 was poured on 10 kg potted soil, mixed evenly
and incubated for 14 days. Three stem cuttings of sweet potato cv Rancing were grown in a pot and placed in the field without shade for 54 days. At seven days after planting, only one best plant was maintained until the end of the experiment.
Chemical fertilization
The chemical fertilizers composed of 200 kg ha-1 of Urea, 100 kg ha-1 of Super Phosphate-36 (SP-36), and 100 kg ha-1 of potassium chloride (KCl) were applied 30 days after planting. The fertilizers were blended evenly and placed in a circular band lay 1 cm below the soil surface at a distance of 2 cm from the stem, while the KCl was placed in another band. All fertilizer holes were then covered with soil. All plants were irrigated with fresh well water in the morning and afternoon to maintain soil humidity since the experiment was conducted in the dry season.
Plant and soil parameters measurement
All parameters were measured at the end of the vegetative stage of the plant at 54 days after planting, including vine length, branches number, root length and volume, fresh and dry weight of shoot and roots, Pb in soil and root, and Azotobacter count sweet potato rhizosphere. The shoots were weighed directly after cutting off from the intact plant. The roots and shoots were put in the paper bag separately, stored at 70 oC for 48 hours in the oven, and then transferred into the desiccator for 20 minutes before weighing. In order to calculate root volume, the individual root was soaked in 100 mL water in a graduated cylinder with an accuracy of 1 mL. The root volume was calculated by reducing the water volume after the roots soaking with 100 mL. Total lead in soil and roots was measured using Atomic Adsorption Spectrophotometer (AAS)
after sample extraction with 1 mL of perchloric acid p.a. and 5 mL of nitric acid p.a. (Sulaeman et al., 2005).
The population of total Azotobacter in the rhizosphere were counted by serial dilution plate method (Ben-Davis and Davidson, 2014). The soil was subjected to serial dilution using NaCl 0.85%. A total of 0.5 mL of soil suspension from 10-5 dilution was poured into a sterilizer 8-cm Petri dish and mixed with Ashby’s agar at 40 oC. The plates were stored at 30 oC for two days until the colony appeared on the surface of Ashby’s agar. The Azotobacter population in soil was calculated by dividing the colony number by the dilution level.
Statistical analysis
All data taken from the pot experiment were analyzed by two-way analysis of variance (ANOVA) to test the significance of the main factors (NFB inoculation and Pb contamination) and their interaction. In order to compare the treatment’s mean difference, the Least Significant Different (LSD) test was performed at p≤0.05. The statistical analysis has been carried out by Statistical Software of Minitab version 18.
Results
Lead resistance of Azotobacter isolates
In this study, all Azotobacter strains showed a one-log decrease in cell density in broth with 1 mg L-1 Pb compared to the control (Table 1). The cell count of azv3 in broth containing 10 mg L-1 Pb was clearly decreased, while that of azv5 was increased.
Azotobacter Azv4 and A. chroococcum showed higher tolerance 100 mg L-1 Pb in N-free broth than other isolates, even though both strains have not been isolated from Pb-uncontaminated soil.
Table 1. Cell count of various isolates of Azotobacter in N-free Ashby’s mannitol broth contaminated with lead.
Pb Azotobacter count (Log10 of CFU mL-1)
(mg L-1) Azv1 Azv2 Azv3 Azv4 Azv5 AC*
0 7.41 ± 0.03 7.33 ± 0.11 7.22 ± 0.12 7.07 ± 0.09 7.17 ± 0.06 7.39 ± 0.11 1 6.77 ± 0.68 6.58 ± 0.07 6.20 ± 0.06 6.82 ± 0.20 6.84 ± 0.19 6.75 ± 0.30 10 6.69 ± 0.08 6.64 ± 0.34 5.98 ± 0.62 6.66 ± 0.18 7.19 ± 0.23 6.66 ± 0.24 100 3.43 ± 0.11 6.06 ± 0.18 5.33 ± 0.50 6.65 ± 0.13 5.53 ± 0.04 7.24 ± 0.14 The values ± SD are the mean of four independent replications, *Azotobacter chroococcum.
Soil contamination by heavy metal cause metabolic stress on bacteria, and it leads to building resistance mechanisms to avoid metal toxicity (Prabhakaran et al., 2016). Nitrogen-fixing Azotobacter has specific detoxification mechanisms, mainly EPS-associated mechanisms for metal extracellular sequestration (Rasulov et al., 2013; Dhevagi et al., 2021). Naturally, Azotobacter synthesizes the EPS as a protectant for nitrogenase by limiting the oxygen flow to the cells (Gauri et al., 2012). Many research findings have demonstrated that heavy-metal resistance and toxicity in beneficial soil microbes are dependent on bacterial
species. Novel in-vitro experiments showed that Azotobacter enables tolerance of 2,000 mg L-1 Pb by excreting EPS and melanin pigment (Rizvi et al., 2019). However, lead tolerance by Azotobacter from Pb-uncontaminated soil has not been widely reported.
Greenhouse experiment
Lead concentration in soil and root
The effect of Azotobacter on Pb accumulation in soil and roots depended on Pb content in soil (Table 2 and Table 3). The lead had not been detected in soil without
Open Access 4275 contamination, but regardless of Azotobacter
inoculation, Pb soil was significantly increased by Pb contamination (Table 2). Higher Pb was found in Pb- contaminated soil without inoculation. The results showed that Azv4 inoculation caused lesser Pb content
in soil with both concentrations of Pb contamination compared to A. chroococcum (AC). In soil with 27.04 mg kg-1 and 37.02 mg kg-1 Pb, the content of Pb soil after Azv4 inoculation was 5.7% and 14.8% less than in AC inoculation, respectively.
Table 2. Interaction effect of Pb contamination and Azotobacter inoculation on Pb in the soil (mg kg-1) after growing sweet potatoes for 54 days.
Azotobacter Pb in soil (mg kg-1) with various Pb content (mg kg-1)
Inoculation Control (17.74) 27.04 37.02
Control 0.0 ± 0.000 a
A 26.0 ± 2.408 c
B 31.7 ± 1.935 c
C
Azv4 0.0 ± 0.000 a
A 17.7 ± 2.495 a
B 16.6 ± 0.837 a
B
AC* 0.0 ± 0.036 a
A
21.0 ± 3.033 b B
19.5 ± 2.320 b B
Values ± SD with the same letter were not significantly different based on LSD test at p ≤ 0.05. Capital letters compared the value in columns, and low case letters compared the value in rows. *A. chroococcum.
Roots grown in uncontaminated soil were free from Pb, but the significant increase of Pb in roots of sweet potatoes grown in both rates of Pb contamination was evidenced (Table 3). Azotobacter Azv4 inoculation caused less Pb in roots than A. choroococcum; the lowest Pb in roots was found in the sweet potato grown in higher rate Pb-contaminated soil with Azv4 inoculation. The Pb root reduction in soil with 37 mg kg-1 Pb by Azv4 isolate was 79.6% and 30%
compared to control and A. chroococcum inoculation, respectively.
Azotobacter population
The interaction effect between different levels of Pb and different Azotobacter strains in N-free broth was significant (Table 4). The Azotobacter population in the control soil and Azv4-inoculated soil significantly increased when the soil was treated with two rates of PbCl2. Statistically, the highest Azotobacter count was shown in the rhizosphere of uninoculated plants grown in soil with 27.04 mg kg-1 and 37.02 mg kg-1 Pb, as well as Azv4-inoculated plants with 37 mg kg-1 Pb.
Table 3. Interaction effect of Pb contamination and Azotobacter inoculation on Pb in roots (mg kg-1) of 54-day old sweet potatoes.
Azotobacter Pb in roots (mg kg-1) with various Pb content (mg kg-1)
Inoculation Control (17.74) 27.04 37.02
Control 0.0 ± 0.000 a
A 45.8 ± 6.385 b
B 51.6 ± 2.708 c
C
Azv4 0.0 ± 0.005 a
A 8.8 ± 6.073 a
B 10.5 ± 1.410 a
B
AC* 0.0 ± 0.008 a
A 12.7 ± 1.970 a
B 15.0 ± 2.427 b
B
Values ± SD with the same letter were not significantly different based on LSD test at p ≤ 0.05. Capital letters compared the value in columns, and low case letters compared the value in rows. *A. chroococcum.
Table 4. Interaction effect of Pb contamination and Azotobacter inoculation on Azotobacter count (104 CFU g-1) in the rhizosphere of 54-days old sweet potatoes.
Azotobacter Population of Azotobacter (104 CFU g-1) with various Pb content (mg kg-1)
inoculation Control (17.74) 27.04 37.02
Control 22.4 ± 12.137 a
A 24.1 ± 9.820 a
A 43.5 ± 13.072 ab
B
Azv4 46.2 ± 16.947 b
B 30.6 ± 5.401 ab
A 30.9 ± 4.037 a
A
AC* 37.3 ± 5.574 b
A 41.2 ±14.725 b
A 45.5 ± 13.996 b
A
Values ± SD with the same letter were not significantly different based on LSD test at p≤0.05. Capital letters compared the value in columns, and low case letters compared the value in rows. *A. chroococcum.
Open Access 4276 Despite the isolation procedure that cannot distinguish
the indigenous and exogenous Azotobacter, Table 4 verified that indigenous Azotobacter might be dominant in soil with or without inoculation. The weak viability of both Azotobacter isolates after inoculation is considered.
Plant growth parameter
Based on ANOVA, the effect of Azotobacter inoculation on plant growth parameter did not determine by Pb content in the soil. Both Azotobacter and Pb did not influence vine length (Figures 1a and 1b) but changed branch numbers (Figures 1c and 1d).
Azotobacter inoculation increased branch number significantly (Figure 1c), but the addition of 27.04 mg kg-1 and 37.02 mg kg-1 Pb reduced branch number by 19.7% and 16.8%, respectively (Figure 1d). In the current study, Azotobacter affected root lengths and volume. Plants with Azotobacter Azv4 and A.
chroococcum had 47 % and 33% higher root lengths compared to the control (Figure 2a). The Azv4 inoculation significantly increased root volume by 17% compared to the A. chroococcum. The increase in soil Pb content did not affect both parameters; there was a slight increase of both traits when plants were grown in Pb-contaminated soil (Figures 2b and 2d),
but it was not significantly different. Each isolate increased shoot and root dry weight but did not cause any change in the shoot to root ratio (Table 5). In contrast, Pb contamination in soil did not influence shoot dry weight but decreased root weight up to 53.1%, which possibly led to the increase in S/R. Soil contamination with 37 mg kg-1 Pb caused R/S increment of 127.7% compared to the control treatment.
The results showed that Pb contamination led to the reduced growth of roots. The experiment verified that Azotobacter Azv4 was more effective in increasing plant growth regardless of the Pb content in the soil.
Discussion
In this current study, Pb was transported to the shoots even though the soil pH was slightly acid. This result was opposite to the general knowledge that the bioavailability of Pb was limited by neutral pH (Yan et al. (2017). It is possible that NPK and organic fertilizers applied to all treatments induced the Pb uptake, which agreed with the increase of certain heavy metal availability (Wierzbowska et al., 2018).
(a) (b)
(c) (d)
Figure 1. Main effect of Azotobacter isolates inoculation and Pb contamination rate on vine length (a and b) and branch number (c and d). Error bars represent the standard error of the mean. Different letters indicate
significantly different based on the LSD test (p≤0.05). AC is A. chroococcum.
a
a
a
0 10 20 30 40 50 60
Control AzV4 AC
Vine lenght (cm)
Azotobacter inoculation
a a
a
0 10 20 30 40 50
Control 27.04 37.02
Vine lenght (cm)
Pb contamination (mg kg-1)
0 5 10 15 20 25 30
Control Azv4 AC
Branch number
Azotobacter inoculation
a a a
0 5 10 15 20 25 30
Control 27.04 37.02
Branch number
Pb contamination (mg kg-1)
Open Access 4277
(a) (b)
(c) (d)
Figure 2. Main effect of Azotobacter isolates inoculation and Pb contamination rate on roots length (a and b) and roots volume (c and d). Error bars represent the standard error of the mean. Different letters indicate significantly
different based on the LSD test (p≤0.05). AC is A. chroococcum.
Table 5. Main effect of Azotobacter inoculation and Pb contamination rate in soil on plant biomass.
Treatments Dry weight (g) S/R
Shoot Root
Azotobacter Inoculation
Control 10.6 ± 2.589 c 1.88 ± 0.67c 7.50 ± 3.18 a
Azv4 22.9 ± 2.085 a 6.27 ± 1.43a 4.40 ± 1.02 a
AC 19.0 ± 4.084 b 3.79 ± 1.68 b 7.55 ± 3.44 a
Pb Contamination
Control 18.0 ± 5.605 a 5.42 ± 2.05 a 4.21 ± 1.86 b
27.04 mg kg-1 16.8 ± 5.880 a 3.99 ± 1.72 b 5.65 ± 2.10 b
37.02 mg kg-1 17.7 ± 6.744 a 2.54 ± 1.95 c 9.59 ± 3.84 a
Values ± SD with the same letter in columns were not significantly different based on LSD test at p ≤ 0.05.
In general, optimal N fertilization increase the distribution of roots and promote the depletion zone, and the uptake of Pb Azotobacter used in this experiment enabled to produce organic matter, which is the source of soil acidity. All the pots treated with the same dose of Urea fertilizer reduced the soil pH, as described by Shetty et al. (2019). However, the soil pH was not measured at the end of the experiment; it is possible the pH was reduced due to Urea application (Lungu and Dynoodt, 2008) and then facilitated Pb
mobilization. Lower total Pb was observed in soil with Azotobacter in any Pb contamination rate verified the Azotobacter mobilized the metal. Available Pb might be uptake by roots, leached out from soil in perforated pots, or elsewhere that cannot be extracted. The heavy metal mobilization by soil bacteria through EPS-Pb ligan formation has been described (Liu et al., 2020);
The EPS of Azotobacter is a bounded EPS on the cell surface. The Pb removal in this study agrees with the role of bounded-EPS in Pb adsorption from solution a
c b
0 10 20 30 40
Control Azv4 AC
Roots lenght (cm)
Azotobacter inoculation
a a a
0 5 10 15 20 25 30 35
Control 27.04 37.02
Roots lenght (cm)
Pb contamination (mg kg-1)
a
c b
0 2 4 6 8 10 12 14 16
Control Azv4 AC
Roots volume (mL
Aotobacter inoculation
a a
a
0 2 4 6 8 10 12 14
Control 27.04 37.02
Roots voume (mL)
Pb contamination (mg kg-1)
Open Access 4278 with a low concentration of Pb (Li et al., 2020). The
EPS of Azotobacter possibly promoted the Pb mobilization by co-migration as described for the divalent metal in saturated porous media (Wu et al., 2021). The result showed that higher Pb in control soil caused Pb uptake increment in roots. Both Azotobacter strain reduced the Pb uptake compared to that in the control. The Azotobacter Azv4 was more prominent in reducing Pb uptake in roots. The different ability to decrease Pb uptake by Azv4 and A. chroococcum could be due to the variation of composition and quantity of EPS of the heavy metal-contaminated condition of Azotobacter (Hindersah, 2015; Hindersah et al., 2017).
Azotobacter promotes branch number, shoot and root dry weight because N2-fixing mechanisms might provide available nitrate in the soil. Phytohormone production by Azotobacter regulates cell development and plant growth. Moreover, the ability of Azotobacter to increase P availability in soil has been reported elsewhere. Nitrogen is essential for plant growth, development and even reproduction. Nitrogen is a major component of chlorophyll, an important substance in photosynthesis by which sunlight energy convert water and carbon dioxide into carbohydrate. It is essential for plants since N is a component of amino acids in the protein. A significant correlation between N level in soil and root and shoot of sweet potato was reported by Relente and Asio (2020). The results were also in line with the effect of Azotobacter inoculation on the growth of tuberous plants and their biomass (Hussien and Ghazi, 2016; Abedaboohanah et al., 2020; Hindersah et al., 2021). However, Azotobacter count in the rhizosphere verified that indigenous Azotobacter is considered to have an equal role in promoting plant growth since the population in the control soil -is not different from Azotobacter-treated soil.
In this study, Pb contamination did not affect shoot parameters but reduced the root dry weight even though it had not influenced root length and volume (Fig 2b and 2d). Lower plant growth traits were more significant in a plant grown in higher Pb contamination soil. The decrease in vine length, branch number, root growth and biomass in Pb-contaminated potted soil was possibly caused by reactive oxygen species formation, cell membrane permeability disruption and photosynthetic rate decline (Hadi and Aziz, 2015;
Jalmi et al., 2018; Nas and Ali, 2018). This resulted in higher S/R of plants with higher Pb levels. Lower S/R indicated that plants grown in Pb-contamination soil have more shoot growth than roots.
The research was terminated in the vegetative phase; there is a possibility that yield reduction will occur due to Pb contamination, especially at a higher level of Pb in soil. The findings of this study are interesting because Pb was absorbed by the roots in significant amounts even though the Pb in soils was below the maximum limit; the soil also had a pH close to neutral. The Pb uptake of uncontaminated roots was
very low, slightly below the AAS detection limit (100 µg kg-1). Fortunately, Pb uptake can be reduced by inoculation of Azotobacter rhizobacteria. This research considers to utilize rhizobacteria Azotobacter for reducing Pb levels in soil and roots and increasing sweet potato biomass. Regardless of Pb contamination, it is clear that rhizobacteria Azotobacter increased the biomass of sweet potato and maintained shoot to root ratio.
Conclusion
The population of five Azotobacter isolates in N-free broth was slightly decreased by Pb rate. Among them, Azotobacter Azv4 and A. chroococcum were more resistant to 100 mg L-1 Pb in the broth. In the pot experiment, the interaction effect between Azotobacter inoculation and Pb contamination was significant.
Azv4 inoculation in soil with 27.04 mg kg-1 and 37.02 mg kg-1 Pb caused 5.7% and 14.8% less Pb in soil than A. chroococcum (AC) inoculation, respectively. The effect of Azotobacter inoculation on plant growth and biomass parameter did not depend on the Pb rate in soil. In the current study, Azotobacter application had not influenced the vine length of sweet potatoes. Inoculation of Azotobacter Azv4 was more prominent in increasing branch number and root volume and length than A. chroococcum. Azotobacter Azv4 inoculation resulted in 13% and 17% more root length and root volume, respectively, compared to the A. chroococcum.
Sweet potatoes grown in soil contaminated by Pb at the rate of 27.04 mg kg-1 and 37.02 mg kg-1 had 19.7% and 16.8% less branch number, respectively than a plant grown in uncontaminated soil. Increased soil Pb clearly reduced length and root volume. This study showed shoot and root dry weight increment after Azotobacter inoculation, while decreased root dry weight was recorded in plants grown in contaminated soil. The results verified that Azotobacter did not change the R/S, but an increase of S/R up to approximately 9 was evident in plants grown in soil containing 37 mg kg-1 of Pb. This research supposed that an increase in Pb accumulation in root is evident even though sweet potatoes were grown in slightly Pb-contaminated soil.
Acknowledgements
Some parts of this research were funded by the Faculty of Agriculture, Universitas Singaperbangsa. The authors thank the Soil Biology Laboratory of the Faculty of Agriculture, Universitas Padjadjaran, for providing chemicals and glass wares during microbiological analysis.
References
Abedaboohanah, M., Kareem, A., Al-Tufaili, H., Hayder, A., Munem, A.A., Turk, H.A.M. and Abdel-Hassan, N.M.
2020. Effect of adding biofertilizer Azotobacter and foliar spray with nitrogen on growth parameters of potato
Open Access 4279 plant Solanum tuberosum L. cv-Bellini. International
Journal of Agricultural and Statistics Sciences 15(2):591-594.
AI-Qasi, B.A., Sharqi, M.M. and Faiath, S.E. 2021. Effect of nitrogen fertilizer sources, lead and cadmium pollution on some properties of barley (Hordeum vulgare). IOP Conference Series: Earth and Environmental Science 904:012057, doi:10.1088/1755-1315/904/1/012057.
Al-Hadi, F., Hussain, F., Hussain, M., Sanaullah, Ahmad, A., Ur Rahman, S. and Ali, A. 2014. Phytoextraction of Pb and Cd; the effect of Urea and EDTA on Cannabis sativa growth under metals stress. International Journal of Agronomy and Agricultural Research 5(3):30-39 Alias, A.A. 2013. Heavy metals in sweet potato and its
potential health risk to human. Thesis. Universiti Teknologi MARA, Selangor, Puncak Alam Campus, Faculty of Health Sciences. Available at https://ir.uitm.edu.my/id/eprint/44188/1/44188.PDF Almaz, M.G., Halim, R.A., Martini, M.Y. and Samsuri,
A.W. 2017. Integrated application of poultry manure and chemical fertiliser on soil chemical properties and nutrient uptake of maize and soybean. Malaysian Journal of Soil Science 21:13-28.
Azzi, V., El Samrani, A., Lartiges, B., Kobeissi, A., Kanso, A. and El-Samrani, A.G. 2017. Trace metals in phosphate fertilizers used in eastern Mediterranean Countries. CLEAN - Soil, Air, Water 45(1):1500988, doi:10.1002/clen.201500988.
Ben-David, A. and Davidson, C.E. 2014. Estimation method for serial dilution experiments. Journal of Microbiological Methods 107:214-221, doi:10.1016/j.mimet.2014.08.023.
Benson, N. 2014. Trace metals levels in inorganic fertilizers commercially available in Nigeria. Journal of Scientific Research and Reports 3(4):610-620, doi:10.9734/JSRR/2014/7465.
Dhevagi, P., Priyatharshinis, Ramya, A. and Sudhakaran, M.
2021. Biosorption of lead ions by exopolysaccharide producing Azotobacter sp. Journal of Environmental Biology 42(1):40-50, doi:10.22438/jeb/42/1/MRN-1231.
Gauri, S.S., Mandal, S.M. and Pati, B.R.. 2012. Impact of Azotobacter exopolysaccharides on sustainable agriculture. Applied Microbiology and Biotechnology 95:331-338, doi:10.1007/s00253-012-4159-0,
Hadi, F. and Aziz, T. 2015. A mini review on lead (Pb) toxicity in plants. Journal of Biology and Life Science 6(2):91-101, doi:10.5296/jbls.v6i2.7152
Handleym, H.K., Blichert-Toft, J., Gertisser, R., Macpherson, C.G., Turner, S.P., Zaennudin, A. and Abdurrachman, M. 2014. Insights from Pb and O isotopes into along-arc variations in subduction inputs and crustal assimilation for volcanic rocks in Java, Sunda Arc, Indonesia. Geochimica et Cosmochimica Acta 139:205-226, doi:10.1016/j.gca.2014.04.025.
Hanyabui, E., Apori, S.O., Frimpong, K.A., Atiah, K., Abindaw, T., Ali, M., Asiamah, J.Y. and Byalebeka, J.
2020. Phosphorus sorption in tropical soils. AIMS Agriculture and Food 5(4):599-616, doi:10.3934/agrfood.2020.4.599.
Hindersah, R. 2015. Growth and composition of exopolysaccharides of nitrogen-fixing bacteria Azotobacter spp. on media containing cadmium.
Proceedings of the Indonesian Biodiversity National
Seminar 1(7):1644-1648,
doi:10.13057/psnmbi/m010718 (in Indonesian).
Hindersah, R., Kamaluddin, N.N., Banerjee, S., Sumanta, S.
and S. Sarkar. 2020. Role and perspective of Azotobacter
in crop production. Sains Tanah-Journal of Soil Science
and Agroclimate 17(2):170-179,
doi:10.20961/stjssa.v17i2.45130.
Hindersah, R., Karuniawan, A. and Apriliana, A. 2021.
Reducing chemical fertilizer in sweet potato cultivation by using mixed biofertilizer. Iraqi Journal of Agricultural Sciences 52(4):1031-1038, doi:10.36103/ijas.v52i4.1414.
Hindersah, R., Mulyani, O. and Osok, R. 2017. Proliferation and exopolysaccharide production of Azotobacter in the presence of mercury. Biodiversity Journal 8(1):21-26.
Holz, M., Zarebanadkouki, M., Kuzyakov, Y., Pausch, Y.
and Carminati, A. 2018. Root hairs increase rhizosphere extension and carbon input to soil. Annals of Botany 121(1):61-69, doi:10.1093/aob/mcx127.
Hussien, H. and Ghazi, A.A. 2016. Effect of biomineral fertilization on sweet potato productivity and quality.
Proceeding of 1st International Conference of Applied
Microbiology pp 21-35,
doi:10.21608/mjss.2016.176576.
Jain, S., Muneer, S., Guerriero, G., Liu, S., Vishwakarma, K., Chauhan, D.K., Dubey, N.K., Tripathi, D.K. and Sharma, S. 2018. Tracing the role of plant proteins in the response to metal toxicity: A comprehensive review.
Plant Signal Behavior 13(9):e1507401, doi:15592324.2018.1507401.
Jalmi, S.K., Bhagat, P.K., Verma, D., Noryang, S., Tayyeba, S., Singh, K., Sharma, D. and Sinha, A.K. 2018.
Traversing the links between heavy metal stress and plant signaling. Frontiers in Plant Science 9:12, doi:10.3389/fpls.2018.00012.
Keran, H., Odobašić, A., Ćatić, S., Salkić, M., Bratovčić, A.
and Šestan, I. 2008. The influence of different soil conditions to the lead bioavailability to plants. 12th International Research/Expert Conference on ”Trends in the Development of Machinery and Associated Technology”, Istanbul, Turkey, 26-30 August 2008.
Kratz, S., Schick, J. and Schnug, E. 2016. Trace elements in rock phosphates and P containing mineral and organo- mineral fertilizers sold in Germany. Science of the Total
Environment 542:1013-1019,
doi:10.1016/j.scitotenv.2015.08.046.
Kuntal Shah, A.U., Mankad, M. and Reddy, N. 2017. Lead accumulation and its effects on growth and biochemical parameters in Tagetes erecta L. International Journal of Life Science and Scientific Research 3(4):1142-1147, doi:10.21276/ijlssr.2017.3.4.7.
Li, N., Liu, J., Yang, R. and Wu, L. 2020. Distribution, characteristics of extracellular polymeric substances of Phanerochaete chrysosporium under lead ion stress and the influence on Pb removal. Scientific Reports 10:17633, doi:10.1038/s41598-020-74983-0.
Liu, H., Li, P., Wang, H., Qing, C., Tan, T., Shi, B., Zhang, G., Jiang, Z., Wang, Y. and Hasan, S.Z. 2020. Arsenic mobilization affected by extracellular polymeric substances (EPS) of the dissimilatory iron-reducing bacteria isolated from high arsenic groundwater. Science of the Total Environment 735(1):139501, doi:10.1016/j.scitotenv.2020.139501.
Luis, G., Rubio, C., Gutiérrez, A.J., González-Weller, D. and Hardisson, C.R.A. 2014. Evaluation of metals in several varieties of sweet potatoes (Ipomoea batatas L.):
Comparative study. Environmental Monitoring and Assessment 186(1):433-440, doi:10.1007/s10661-013- 3388-8.
Lungu, O.I. and Dynoodt, R.F.P. 2008. Acidification from long-term use of urea and its effect on selected soil
Open Access 4280 properties. African Journal of Food Agriculture
Nutrition and Development 8(1):63-76.
Ministry of State for Population and Environment Republic of Indonesia and Dalhousie University Canada. 1992.
Environmental Management in Indonesia. Report on Soil Quality Standards for Indonesia (interim report).
Mukhtar, H., Bashir, H. and Nawaz, A. 2018. Optimization of growth conditions for Azotobacter species and their use as biofertilizer. Journal of Bacteriology and
Mycology 6(5):274-278,
doi:10.15406/jbmoa.2018.06.00217.
Nas, F.S. and Ali, M. 2018. The effect of lead on plants in terms of growing and biochemical parameters: A review.
MOJ Ecology and Environmental Science 3(4):265-268, doi:10.15406/mojes.2018.03.00098.
Pembere, A.M.S. 2018. Heavy Metal Levels in Food Crops and Soils from Western Kenya Paperback-16 July 2018.
Lambert Academic Publishing. Chisinau, MD-2012, Republic of Moldova. pp 96.
PPT. 1993. Soil Research Center Regulation. Regulation number 59. Soil Research Center, Bogor, Indonesia (in Indonesian).
Prabhakaran, P., Ashraf, M.A., Aqma, W.S. and Noor, W.M.
2016. Microbial stress response to heavy metal in the environment. RSC Advances 6:109862-109877, doi:10.1039/C6RA10966G.
Rasulov, B.A., Yili, A. and Aisa, H.A. 2013. Biosorption of metal ions by exopolysaccharide produced by Azotobacter chroococcum XU1. Journal of Environmental Protection 4(9):989-993, doi:10.4236/jep.2013.49114.
Relente, F.A.C. and Asio, L.G. 2020. Nitrogen application improved the growth and yield performance of sweet potato (Ipomoea batatas (L) Lam.). Annlas of Tropical Research 42(1):45-55, doi:10.32945/atr4214.2020.
Rizvi, A., Ahmed, B., Zaidi, A. and Khan, M.S. 2019.
Bioreduction of toxicity influenced by bioactive molecules secreted under metal stress by Azotobacter chroococcum. Ecotoxicity 28(3): 302-322, doi:10.1007/s10646-019-02023-3.
Shetty, P., Acharya, C. and Veeresh, N. 2019. Effect of urea fertilizer on the biochemical characteristics of soil.
International Journal of Applied Science and
Biotechnology 7(4):414-420,
doi:10.3126/ijasbt.v7i4.26778.
Shi, Z., Di Toro, D.M., Allen, H.E and Sparks, D.L. 2013. A general model for kinetics of heavy metal adsorption and desorption on soils. Environmental Science and Technology 47(8):3761-3767, doi:10.1021/es304524p.
Sulaeman, Suparto and Eviati. 2005. Technical Guidelines:
Chemical Analysis of Soil, Plants, Water, and Fertilizers.
Indonesian Soil Research Institute. 234p ISBN 978-602- 8039-21-5 (in Indonesian).
Upadhyay, S.K., Kumar, N., Singh, V. K. and Singh, A.
2015. Isolation, characterization and morphological study of Azotobacter isolates. Journal of Applied and Natural Science 7(2 SE): 984-990, doi:10.31018/jans.v7i2.718.
Upadhyay, S.K., Srivastava, A.K., Rajput, V.D., Chauhan, P.K., Bhojiya, A.A., Jain, D., Chaubey, G., Dwivedi, P., Sharma, B. and Minkina, T. 2022. Root exudates:
Mechanistic insight of plant growth promoting rhizobacteria for sustainable crop production. Frontiers
in Microbiology 13:916488,
doi:10.3389/fmicb.2022.916488.
Van Oosten, M.J., Di Stasio, E., Cirillo, V., Silletti, S., Ventorino, V., Pepe, O., Raimondi, G. and Maggio, A.
2018. Root inoculation with Azotobacter chroococcum 76A enhances tomato plants adaptation to salt stress under low N conditions. BMC Plant Biology 18:205, doi:10.1186/s12870-018-1411-5.
Wei, B., Yu, J., Cao, Z., Meng, M., Yang, L. and Chen, Q.
2020. The availability and accumulation of heavy metals in greenhouse soils associated with intensive fertilizer application. International Journal of Environmental Research and Public Health 17:5359, doi:10.3390/ijerph17155359.
Wierzbowska, J., Kovačik, P., Sienkiewicz, S., Krzebietke, S. and Bowszys, T. 2018. Determination of heavy metals and their availability to plants in soil fertilized with different waste substances. Environmental and Monitoring Assessment 190:567, doi:10.1007/s10661- 018-6941-7.
Williams, L.E., Pittman, J.K. and Hall, J.L. 2000. Emerging mechanisms for heavy metal transport in plants.
Biochimica et Biophysica Acta-Biomembranes 1465(1- 2):104-126, doi:10.1016/S0005-2736(00)00133-4.
Wu, Y., Li, Z., Yang, Y., Purchase, D., Lu, Y. and Dai Z.
2021. Extracellular polymeric substances facilitate the adsorption and migration of Cu2+ and Cd2+ in saturated porous media. Biomolecules 11(11):1715, doi:10.3390/biom11111715.
Yan, K., Dong, Z., Wijayawardena, M.A.A., Liu, Y. and Naidu, R. 2017. Measurement of soil lead bioavailability and influence of soil types and properties: A review.
Chemosphere 184(October):27-42,
doi:10.1016/j.chemosphere.2017.05.143.
