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Article

Utilization of Palm Oil Midrib Biochar as Soil Amendment with a Newly Isolated Bacillus sp. SM11 for Growth Enhancement and Nitrate Reduction in Romaine Lettuce (Lactuca sativa L. var. longifolia)

by
Sukhan Rattanaloeadnusorn
,
Nopparat Buddhakala
and
Thanasak Lomthong
*
Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi, Pathum Thani 12110, Thailand
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(10), 1986; https://doi.org/10.3390/agriculture13101986
Submission received: 22 September 2023 / Revised: 10 October 2023 / Accepted: 12 October 2023 / Published: 12 October 2023
(This article belongs to the Section Agricultural Soils)
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Abstract

:
Biochar is a carbon-rich material that enhances nutrient availability, soil quality, and microbial activity, improving plant growth and crop productivity. In this study, the palm oil midrib biochar (POMB) was used as a soil conditioner to improve the growth of romaine lettuce (Lactuca sativa L. var. longifolia), together with a biofertilizer containing a newly isolated bacterial strain SM11. The newly isolated SM11 was closely related to Bacillus siamensis, with 99.77% similarity based on 16s rRNA gene sequence analysis. POMB treatment improved the fresh weight of romaine lettuce by up to 181.33 ± 1.15 g plant−1, which is equivalent to 160.27% growth enhancement compared to the control without POMB. By comparison, POMB treatment with a biofertilizer containing SM11 increased fresh weight to 275.67 ± 11.59 g plant−1 with a growth enhancement of 295.68%. The addition of SM11 biofertilizer also protected against disease during cultivation. The addition of POMB reduced nitrate accumulation in romaine lettuce from 631.38 ± 0.36 to 223.31 ± 0.20 mg kg−1 by 59.08% compared to the control. This study suggests a way to manage agricultural waste from local palm oil plantations, thereby reducing waste accumulation and adding value to palm oil waste by-products for agricultural benefit through a biotechnological process.

1. Introduction

Thailand is the third-largest global producer of palm oil for use in food, cosmetics, and biofuels [1]. Palm oil production generates biomass solid waste as palm oil midribs at 14.2 million tons per hectare every year [2]. The palm oil midrib, also known as the palm leaf stem or rachis, is an essential part of palm leaves and is rich in cellulose, hemicellulose, and lignin [2,3]. Recently, the palm oil midrib has been utilized to produce activated carbon for the adsorption of dyes from wastewater generated by the textile industry [4]. However, few studies have reported on the use of biochar for soil amendment.
“Biochar” refers to the carbon-rich solid material created during biomass pyrolysis or thermal degradation [5]. Biochar has been shown to enhance water retention, nutrient availability, and microbial activity in the soil, leading to improved plant growth and crop productivity [6,7]. Biochar has also shown potential benefits in promoting plant growth using various mechanisms, such as enhanced nutrient retention due to a high cation exchange capacity (CEC) that attracts and retains nutrients in the soil and prevents nutrient leaching [6]. Biochar also enhances soil fertility by increasing the content of organic matter and improving soil structure [8]. According to Carter et al. [9], biochar increases the pH by 0.5 to 1.0 units, and nutrients are directly available through the solubilization of ash in solid biochar residue. Other nutrients may also become available through microbial use. Carter et al. [9] reported that the application of biochar in addition to fertilized soil improved the growth of lettuce (Lactuca sativa) and Chinese cabbage (Brassica chinensis) by increasing the final biomass, root biomass, plant height, and number of leaves across all cropping cycles when compared to the treatment without biochar. More than 85% of studies concluded that biochar might decrease plant disease by modifying beneficial microbial populations, nutrient availability, and the hormesis effects of phytohormones obtained from biochar. The processes included in these mechanisms are nutrition supply and the induced resistance of plants [10]. In this study, palm oil midrib biomass was used as a substrate for biochar production, providing a sustainable use for this waste biomass to improve crop productivity.
Romaine lettuce (Lactuca sativa L. var. longifolia), or cos lettuce, is popular in salads and sandwiches and also provides dietary fiber, fatty acids, mineral contents, and various antioxidants [11]. However, romaine lettuce, like other leafy green vegetables, is prone to nitrate (NO3−) accumulation under certain conditions [12,13]. Nitrate accumulation is a concern because high levels of nitrates in leafy greens can harm human health. However, with proper cultivation practices, the risk of nitrate accumulation can be minimized [14,15]. According to Bian et al. [14], plant roots mostly absorb nitrate from the soil, and the overfertilization of nitrogen, particularly nitrate fertilizer, has been utilized in crop production to increase crop yield. It has been observed that throughout cultivation, the vegetable leaf accumulates high quantities of nitrates totaling more than 700 mg kg−1, which is dangerous for human health and can result in gastric cancer and methemoglobinemia in young children [14]. Nitrate accumulation in plants can be reduced through the addition of biochar to the soil, which stimulates denitrification, resulting in decreased N2O accumulation [16]. The changes in the soil pH when adding biochar also decrease the enzyme activity involved in denitrification and also reduce the accumulation of nitrate in plants.
This study developed biochar from palm oil midrib as a soil amendment to induce growth and nitrate reduction with a newly isolated Bacillus sp. SM11 adds value to agricultural waste and provides the application knowledge of palm oil midrib biochar with a microbial fertilizer for agricultural benefit.

2. Materials and Methods

2.1. Palm Oil Midrib Biochar (POMB) Preparation

Palm oil leaves were obtained from the Bueng Ba Community, Nong Suea District, Pathum Thani Province, Thailand. The obtained palm oil leaves were dried in a solar greenhouse with temperature controlled at 50 °C for 7 days and then separated from the dried midribs for biochar production.
The dried palm oil midribs (100 kg) were subjected to slow pyrolysis in a muffle furnace (88 × 40 × 40 cm3) equipped with a digital temperature regulator at 600 °C under continuous nitrogen (purity-99.9%) purging with a heating rate of 15 °C min−1. The POMB was powdered by an electric grinder and filtered through a 0.2 mm mesh, and the morphological structure and chemical composition were characterized using a Scanning Electron Microscope (SEM) (JEOL, JSM-5410 LV, Tokyo, Japan) and Energy Dispersive X-ray Spectrometer (EDS) (JEOL, Tokyo, Japan) [17].

2.2. Soil and Rice Husk Preparation

The lettuce test soil was obtained at a 0–20 cm depth from arable land in a field experiment at the Bueng Ba Community, Nong Suea District, Pathum Thani Province, Thailand. The collected soil was adjusted to pH 6.5 by adding pumice powder 3% (w/w).
Rice husk was used for microbial fertilizer, and soil media preparation containing less than 10% moisture content was obtained from a local rice mill in Nong Suea District, Pathum Thani Province, Thailand. Rice husk was used in the soil and fertilizer mixture because it is highly porous and light, allowing for air circulation and the retention of water.

2.3. Microorganism and Fertilizer Preparation

The bacterial strain SM11, previously isolated from the lettuce planting field soil, was obtained from the Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi, Pathum Thani, Thailand. The SM11 strain was identified based on 16s rRNA gene sequencing by the Thailand Bioresource Research Center (TBRC), National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand. Genomic DNA was extracted using a Genomic DNA mini kit (Geneaid Biotech Ltd., New Taipei City, Taiwan). The 16s rRNA gene was amplified using the universal primers 20F 5′-GAG TTT GAT CCT GGC TCA G-3′ and 1500R 5′-GTT ACC TTG TTA CGA CTT-3′. The selected sequences were first aligned with the ClustalW program using BioEdit version 7.2.5 software and were used for further phylogenetic analysis with MEGA 11 software [18,19,20,21]. The biochemical characteristics of the SM11 strain were investigated using Vitex-2 Compact® from Biomérieux, France [22].
To prepare the starter culture, the SM11 strain was cultivated in nutrient broth in a 250 mL shaking flask at 30 °C for 72 h, providing an optical density of 0.7 at a wavelength of 600 nm, equivalent to 1 × 107 CFU/mL on a nutrient agar plate.
The microbial fertilizer was prepared by mixing 0.3% (v/w) of the SM11 strain with 0.5% (w/w) molasses, 5.0% (w/w) crushed palm leaves, 2.0% (w/w) salt pan sediments, 0.15% (w/w) rice husk, and 5.0% (w/w) of chicken manure. The moisture content was adjusted to 60% with deionized groundwater, the pH was adjusted to 6.5, and the mixture was incubated for 45 days. Then, the mixture was powdered, filtered through a 0.5 mm mesh, dried at room temperature for 48 h, and used as a biofertilizer for the growth of romaine lettuce.

2.4. Effect of Palm Oil Midrib Biochar on Growth Induction of Romaine Lettuce

Romaine lettuce seeds were obtained from the Breeding and Seed Production Center for Organic Vegetables, Mae Jo University, Thailand, and kept dry until used. The seeds were seeded in a seedling tray (2.0 × 2.0 × 4.0 cm3) containing peat moss (PM) as the planting material for 15 days, which were then used for the experiments.
The experiments were conducted at the Bueng Ba Community, Nong Suea District, Pathum Thani Province, Thailand (14°8′6″ N, 100°49′27″ E) with an average temperature of 31 °C and air humidity of 72%. Four treatments with three replications were performed using a complete randomized design (CRD) to examine the impact of POMB and microbial fertilizer, both individually and in combination, on the growth and nitrate levels of romaine lettuce, as described below.
(T1): Control treatment without adding POMB and a microbial fertilizer was prepared by mixing dried non-amended soil with rice husk in a ratio of 1:3, which was used as the control soil medium.
(T2): POMB treatment was prepared by mixing 3.0% (w/w) biochar with dried non-amended soil and rice husk (ratio 1:3) obtained from the control treatment (T1), using POMB treatment to examine the effect of sole POMB.
(T3): Microbial fertilizer treatment, prepared by adding 0.3% (v/w) of the microbial fertilizer to the dried non-amended soil with rice husk (ratio 1:3) obtained from the control treatment (T1), was used as the soil medium to determine the effect of microbial fertilizer.
(T4): POMB with microbial fertilizer treatment, prepared by adding 0.3% (v/w) of microbial fertilizer to the soil, similar to the T2 treatment, which contained non-amended soil with rice husk (ratio 1:3) and 3.0% (w/w) biochar, was used as the soil medium to investigate the impact of POMB and the microbial fertilizer.
Four soil media were analyzed for their chemical composition, including pH, electrical conductivity (EC), organic matter, macronutrients, and micronutrients, following AOAC (1990) at the analysis unit of the land development regional office, Pathum Thani Province, Thailand.
Next, one of the 15-day-old seedlings of romaine lettuce 3.0 cm in height was separated and transferred to plastic pots (8 × 5 × 11 inches) containing each of the four soil media as described above with 15 replicates per trial and three replicates were sampled at interval times during 5 weeks of cultivation for analysis. The growth phase was five weeks, with deionized groundwater sprayed daily to maintain 60% soil humidity. Samples were taken every week to measure the number of leaves (leaf), the width of leaves (cm), fresh plant weight (g), and disease traces. Growth enhancement of romaine lettuce was calculated for comparison with the control, as shown in Equation (1).
Growth enhancing rate (%) = (Test − Control/Control) × 100

2.5. Nitrate Accumulation Measurement

After planting for five weeks, the samples were analyzed using a spectrophotometric approach similar to that described by Cataldo et al. [23] with slight modification. Two grams of the material were extracted for 30 min at 100 °C, cooled with tap water, filtered, and diluted to a volume of 25 mL with distilled water. A solution of 5% (w/v) salicylic acid-concentrated sulfuric acid and 0.1 mL of the extract were combined. Approximately 20 min later, 9.5 mL of an 8% (w/v) NaOH solution was added, and the absorbance at 410 nm was measured. The nitrate (NO3−) reduction in each experiment was calculated using Equation (2):
Nitrate reduction (%) = (Nitrate Test − Nitrate Control/Nitrate Control) × 100
The total soluble solids (TSS, °Brix) value was used to estimate the sugar contents in samples and referred to the vegetable quality, which was measured by mashing a 10.0 g sample of leaves and using a refractometer (RA-250WE, Kyoto Electronics, Kyoto, Japan).

2.6. Statistical Analysis

Data were collected and analyzed using two methodologies, including descriptive statistics as the mean ± SD and inferential statistics using the R-studio program (Version-RStudio 2022.12.0+353) using p < 0.05, Bartlett’s test, F-Test (One-way ANOVA), and multiple comparisons with least significant difference (LSD) and three replications.

3. Results and Discussion

3.1. Characterization of Palm Oil Midrib Biochar

The POMB was obtained after pyrolysis at 600 °C, with scanning electron micrographs of POMB shown in Figure 1. The POMB structure contained pores 5.0 to 10.0 µm in diameter (Figure 1) that were appropriate for microbial colonization [24]. Thies and Rillig [25] reported that the porous structure of biochar adsorbed soluble organic materials, gases, and inorganic nutrients, creating a highly favorable habitat for microbes such as bacteria, actinomycetes, and arbuscular mycorrhizal fungi to colonize, thrive, and reproduce, while Yang et al. [26] stated that biochar pore structures greater than 5.0 µm increased the diversity and abundance of soil microbes. Moreover, Singh et al. [27] also reported that the presence of numerous macro- and micro-pores on the surface of biochar can be used to treat heavy metal-contaminated soil and improve the water-holding capacity. POMB elemental composition was characterized using EDS, as shown in Table 1. The main component was carbon (C) at 76.26%, with oxygen (O) at 19.55%, and a high carbon content in POMB results indicating a higher purity of biochar. The other elements at less than 2.0% functioned as supplementary nutrients for plant growth promotion, including 1.56% potassium (K), 0.27% phosphorus (P), 0.52% magnesium (Mg), 0.67% calcium, 0.07% silicon (Si), and 0.15% zinc (Zn).

3.2. Identification of Bacterial Fertilizer

The molecular taxonomy of 16S rRNA gene sequences for strain SM11 was closely related to Bacillus siamensis KCTC 13613 (AJVF01000043) with a sequence similarity of 99.77% (Figure 2). B. siamensis is a Gram-positive, endospore-forming, rod-shaped bacterium that can grow at both mesophilic and thermophilic temperatures (25–55 °C) [28]. B. siamensis also showed biocontrol efficiency against tobacco brown spot disease caused by Alternaria alternata and had antimicrobial and plant growth-promoting properties [29]. Hussain and Khan [30] reported that the B. siamensis strain AMU03 acted against soil-borne fungal pathogens of potato tubers, Rhizoctonia solani, and Fusarium oxysporum under in vitro testing. Awan et al. [31] also reported that B. siamensis could reduce cadmium contamination in the soil and improve the growth and antioxidant defense system in wheat plants. The biochemical characteristics of the SM11 strain are shown in Table 2. This strain produced various metabolic enzymes with the ability to utilize different substrates as a good characteristic of bacterial fertilizer.

3.3. Effect of Palm Oil Midrib Biochar on Growth Induction of Romaine Lettuce

The chemical compositions of soil in each treatment are shown in Table 3, with pH values ranging from 5.60 to 6.56. Adding POMB increased the soil’s pH because biochar has an alkaline pH and significantly increased the soil pH by about 0.5 to 1.0 units from non-amendment soil [9,32]. The addition of biochar and a microbial fertilizer increased soil electrical conductivity, organic matter, macronutrients (N, P, K), micronutrients such as calcium, and cation exchange capacity (CEC), which refers to the total number of cations that a soil can hold by 1.5-fold compared to the control (Table 3), thereby enhancing growth.
The results of each condition for the growth of romaine lettuce are shown in Figure 3. The addition of POMB (T2) improved the number of leaves (Figure 3A), width of leaves (Figure 3B), and weight of romaine lettuce (Figure 3C) during cultivation for five weeks, with a growth enhancement of 27.06, 47.39, and 160.27%, respectively, compared to the control (T1) (Figure 3D). The positive effect observed for romaine lettuce growth was probably caused because biochar was reported in previous studies as long-term soil carbon sequestration [33] and has long-lasting impacts on soil fertility, water retention, and nutrient availability by promoting nutrient retention, boosting microbial activity, and reducing soil erosion [34,35,36].
T4 containing POMB and the fertilizer using the SM11 strain showed the highest improvement in romaine lettuce growth with 39.56, 78.99 and 295.68% of growth enhancement for leaf number, leaf width, and the weight of the plant, respectively (Figure 3D). Fresh weight/plant at week 5 was 69.67 ± 1.67, 181.33 ± 1.15, 200.60 ± 11.59, and 275.67 ± 7.29 g for T1 (control), T2 (POMB), T3 (MF), and T4 (POMB+MF), respectively. The results suggest that combining POMB and fertilizer containing the SM11 strain can improve the growth of romaine lettuce more than other treatments.
Adding bacterial fertilizer containing SM11 solely (T3) also enhanced the growth of romaine lettuce but not so much as the combination with POMB (T4). The porous structure of biochar functions as a habitat for bacterial colonization [37]. Lu et al. [38] reported that adding rice straw biochar with Bacillus spp. improved the growth of plant growth-promoting bacteria, which can adsorb the nutrients available for microbial use. In this study, adding Bacillus sp. SM11 to POMB enhanced the growth of romaine lettuce, with the potential for future development as a commercial biochar-based microbial fertilizer. Romaine lettuce growth after cultivation for five weeks under four different treatments is shown in Figure 4.

3.4. Nitrate Accumulation, °Brix and Leaf Spot Disease Measurement

Nitrate accumulation, °Brix, and leaf spot disease in each treatment are shown in Table 4. Nitrate accumulation in T1 was 631.38 ± 0.36 mg kg−1, while T2-containing POMB provided 223.31 ± 0.20 mg kg−1 (Table 4). Wu et al. [16] reported that biochar produced from cattail (Typha latifolia) at 300–500 °C increased the total nitrogen removal by 415% and decreased N2O accumulation by 78% in water treatment by acting as the bioengineer of the electron shuttle and the stimulator of denitrification. In this study, the biochar produced from palm oil midrib waste at 600 °C could reduce nitrate accumulation by up to 59.08 and 64.59% in T2 and T4 treatments, respectively. Jiang et al. [32] reported that adding biochar to soil increased the pH from 4.0 to 6.1 and reduced the amount of NO3 in deteriorated soil from 180 to 20 mg kg−1. The N2O product ratio for denitrification dropped in soil with a relatively high pH due to decreased N2O reductase enzyme activity or enzyme production [32].
Total soluble solids (TSS) or °Brix in the samples found that treatment with the SM11 biofertilizer elevated leaf sugar content because nutrients from the biofertilizer induced increased photosynthesis in romaine lettuce, which promoted sugar metabolism and related enzyme activities [39,40,41,42]. Adding biochar to the fertilizer (T4) also increased the °Brix content to 3.0 °Brix. Lebrun et al. [42] reported that biochar added with manure fertilizer increased the sugar contents in plants by 2.4-fold because the biochar improved soil moisture and reduced nutrient leaching, thereby supporting plant metabolism and the photosynthesis process.
Leaf spot disease was found in T1 and T2 but not in T3 and T4. The results suggest that biofertilizers containing the SM11 strain protect against leaf spot disease during cultivation compared to treatments without SM11 biofertilizers. The leaf spot disease was not observed in T3 due to the fact that the microbial fertilizer with Bacillus sp. SM11 may alter the microbial community in soil, which is competitive to the growth of pathogenic microorganisms [8]. While T4 contained both biochar and microbial fertilizer, leaf spot disease was also not found due to modifying beneficial microbial populations, nutrient content and availability, and the hormesis effects of phytohormones obtained from biochar, as reported by Frenkel et al. [10].
POMB is used for soil amendment with a newly isolated Bacillus sp. SM11 showed enhanced growth and nitrate reduction in romaine lettuce and could be applied for further biochar-based biofertilizer development.

4. Conclusions

POMB was developed as a soil amendment to add value to agricultural waste from local palm oil plantations. POMB’s addition improved the soil and enhanced the growth of romaine lettuce. The addition of POMB increased the weight of romaine lettuce by 160.27% with a reduction in nitrate at 59.08%, while the co-function of POMB with a newly isolated B. siamensis SM11 strain improved growth and nitrate reduction at 295.68% and 64.59%, respectively. The development of biochar with the B. siamensis SM11 strain as a commercial multifunctional fertilizer needs to be investigated in future research. This study added value to palm oil midrib waste biomass for use as a soil amendment material with a novel bacterial strain to improve growth and nitrate reduction for agricultural benefit.

Author Contributions

S.R.: Conceptualization, investigation, writing—original draft preparation. N.B.: Investigation and suggestions. T.L.: data acquisition and analysis, writing review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

We are grateful to the Research Fund of the Ministry of Science and Technology and the Faculty of Science and Technology Rajamangala University of Technology, Thanyaburi, Pathum Thani, Thailand, for financial support (Project No: 2492842).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All relevant data are provided within the manuscript.

Acknowledgments

We also thank Siriwan Kongkaphan from Innofarmbiotec Co., Ltd., Kanchanaburi Province, Pajon Vangchom, Chairman of the Community Enterprise, Nong Suea Organic Garden, Pathum Thani Province, Thailand and Thinnakorn Aromsuk for supplying the substrate and for the use of machine facilities.

Conflicts of Interest

The authors declare no conflict of interest.

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Agriculture 13 01986 g001
Figure 1. Scanning electron micrographs of palm oil midrib biochar. (A) 1000× and (B) 3500×.
Figure 1. Scanning electron micrographs of palm oil midrib biochar. (A) 1000× and (B) 3500×.
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Agriculture 13 01986 g002
Figure 2. Phylogenetic tree of SM11 and related type strains within the genus Bacillus based on partial 16S rRNA gene sequences using the neighbor-joining method algorithm in MEGA 11 software.
Figure 2. Phylogenetic tree of SM11 and related type strains within the genus Bacillus based on partial 16S rRNA gene sequences using the neighbor-joining method algorithm in MEGA 11 software.
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Agriculture 13 01986 g003
Figure 3. Effect of palm oil midrib biochar and microbial fertilizer on growth of romaine lettuce at different cultivation times. (A) Average number of leaves, (B) Average width of leaves, (C) Average weight/plant and (D) Growth-enhancing rate after five weeks of cultivation. Values are averages of three determinations. POMB: palm oil midrib biochar, MF: microbial fertilizer.
Figure 3. Effect of palm oil midrib biochar and microbial fertilizer on growth of romaine lettuce at different cultivation times. (A) Average number of leaves, (B) Average width of leaves, (C) Average weight/plant and (D) Growth-enhancing rate after five weeks of cultivation. Values are averages of three determinations. POMB: palm oil midrib biochar, MF: microbial fertilizer.
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Agriculture 13 01986 g004
Figure 4. Growth of romaine lettuce after cultivation for five weeks. (A) T1, (B) T2, (C) T3 and (D) T4.
Figure 4. Growth of romaine lettuce after cultivation for five weeks. (A) T1, (B) T2, (C) T3 and (D) T4.
Agriculture 13 01986 g004
Table 1. Elemental composition of palm oil midrib biochar.
Table 1. Elemental composition of palm oil midrib biochar.
ElementComposition (%)
C76.26 ± 0.03
O19.55 ± 0.06
P0.27 ± 0.01
K1.56 ± 0.02
Mg0.52 ± 0.01
Ca0.67 ± 0.01
Si0.07 ± 0.00
Mn0.01 ± 0.00
Cu0.18 ± 0.02
Zn0.15 ± 0.01
S0.24 ± 0.01
Table 2. Biochemical characteristics of the SM11 strain using Vitex-2 Compact®.
Table 2. Biochemical characteristics of the SM11 strain using Vitex-2 Compact®.
CharacteristicResultCharacteristicResult
Gram reaction +ve
β-xylosidase +Cyclodextrine−
L-lysine arylamidase−D-mannitol +
L-aspartate arylamidase−D-mannose +
Leucine arylamidase (+)D-galactose−
Phenylalanine arylamidase +D-melezitose−
L-pyrrolidonyl arylamidase−N-acetyl-D-glucosamine−
β-galactosidase−Palatinose +
L-pyrrolidonyl arylamidase +L-rhamnose−
α-galactosidase +β-mannosidase−
Alanine arylamidase−Phosphoryl choline−
Tyrosine arylamidase +Pyruvate−
β-N-acetyl-glucosaminidase−α-glucosidase−
Ala-Phe-Pro arylamidase(−)D-tagatose−
Glycine arylamidase−D-trehalose−
β-glucosidase +Inulin−
Glycogene−D-glucose +
Myo-inositol−D-ribose (−)
Methyl-α-D-glucopyranoside acidification +Putrescine assimilation−
Kanamycin resistance−Growth in 6.5% NaCl +
Methyl-D-xyloside−Oleandomycin resistance−
α-mannosidase−Esculin hydrolysis+
Maltotriose−Polymixin_B resistance−
Note: + = positive reaction; − = negative reaction; (+) = weak–positive reaction; (−) = weak–negative reaction; +ve = Gram positive bacteria.
Table 3. Chemical composition of soil used in the experiment.
Table 3. Chemical composition of soil used in the experiment.
Chemical CompositionTreatment
T1 (Control)T2 (POMB)T3 (MF)T4 (POMB+MF)
pH5.60 ± 0.100 b6.56 ± 0.01 a6.55 ± 0.050 a6.53 ± 0.025 a
EC (dS m−1)0.01 ± 0.006 a4.09 ± 0.015 b4.07 ± 0.023 a4.37 ± 0.047 a
OM (%)2.49 ± 0.360 c3.55 ± 0.050 b3.65 ± 0.101 b4.88 ± 0.068 a
N (%)0.08 ± 0.010 d1.25 ± 0.010 c1.35 ± 0.031 b1.55 ± 0.015 a
P (%)10.02 ± 0.023 d15.30 ± 0.051 c15.69± 0.051 b18.20 ± 0.100 a
K (cmol kg−1)521.70 ± 0.100 d582.77 ± 0.252 c612.11 ± 0.161 b627.53 ± 0.208 a
Mg (cmol kg−1)123.03 ± 0.006 d302.04 ± 0.015 c306.05 ± 0.084 b337.78 ±1.130 b
Ca (cmol kg−1)1450.06 ± 0.006 d1501.05 ± 0.015 c1512.02 ± 0.016 a1511.20 ± 0.100 b
S (%)0.59 ± 0.006 b1.54 ± 0.012 a1.55 ± 0.006 a1.543 ± 0.002 a
Na (%)0.12 ± 0.006 a0.09 ± 0.012 b0.09 ± 0.010 b0.08 ± 0.010 b
Fe (mg kg−1)0.12 ± 0.029 b0.15 ± 0.006 a0.16 ± 0.010 a0.163 ± 0.006 a
Mn (mg kg−1)10.53 ± 0.058 d19.45 ± 0.132 c42.60 ± 0.361 b59.27 ± 0.252 a
Zn (mg kg−1)140.33 ± 0.577 b156.00 ± 1.000 a156.50 ± 0.200 a156.33 ± 0.289 a
Cu (mg kg−1)100.16 ± 0.058 b116.27 ± 0.058 a116.27 ± 0.058 a116.27 ± 0.058 a
As (mg kg−1)143.91± 0.170 a143.90± 0.608 a142.00 ± 0.500 b142.16 ± 0.289 b
Cd (mg kg−1)134.10 ± 0.100 a134.20 ± 0.100 a133.16 ± 0.115 b132.35 ± 0.198 c
Cr (mg kg−1)140.23 ± 0.58 a140.30 ± 0.127 a134.26 ± 0.153 b134.23 ± 0.208 b
Pb (mg kg−1)116.18 ± 0.020 a116.18 ± 0.025 a115.08 ± 0.021 b115.18 ± 0.161 b
Hg (mg kg−1)0.13 ± 0.031 b0.15 ± 0.003 a0.15 ± 0.003 a0.15 ± 0.008 a
Note: Values are averages of three determinations. Different letters (a,b,c,d) among the columns are statistically different at p < 0.05 via LSD analysis.
Table 4. Nitrate accumulation in romaine lettuce after cultivation for five weeks.
Table 4. Nitrate accumulation in romaine lettuce after cultivation for five weeks.
Treatment°BrixNO3− Accumulation
(mg kg−1)		%NO3−
reduction		Leaf Spot Disease
T1 (Control)0.96 ± 0.029 c631.38 ± 0.36 a1.00+++
T2 (POMB)0.98 ± 0.029 c223.31 ± 0.20 d59.08+++
T3 (MF)2.05 ± 0.123 b234.10 ± 0.90 c62.92+
T4 (POMB+MF)3.01 ± 0.161 a258.30 ± 0.058 b64.59−
Note: Values are averages of three determinations. Different letters (a, b, c, d) among the columns are statistically different at p < 0.05 via LSD analysis. − = No spot disease was observed. + = Spot disease was found at 1 point per leaf. ++ = Spot disease was found at 2–3 points per leaf. +++ = Spot disease was found at more than 3 points per leaf.
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MDPI and ACS Style

Rattanaloeadnusorn, S.; Buddhakala, N.; Lomthong, T. Utilization of Palm Oil Midrib Biochar as Soil Amendment with a Newly Isolated Bacillus sp. SM11 for Growth Enhancement and Nitrate Reduction in Romaine Lettuce (Lactuca sativa L. var. longifolia). Agriculture 2023, 13, 1986. https://doi.org/10.3390/agriculture13101986

AMA Style

Rattanaloeadnusorn S, Buddhakala N, Lomthong T. Utilization of Palm Oil Midrib Biochar as Soil Amendment with a Newly Isolated Bacillus sp. SM11 for Growth Enhancement and Nitrate Reduction in Romaine Lettuce (Lactuca sativa L. var. longifolia). Agriculture. 2023; 13(10):1986. https://doi.org/10.3390/agriculture13101986

Chicago/Turabian Style

Rattanaloeadnusorn, Sukhan, Nopparat Buddhakala, and Thanasak Lomthong. 2023. "Utilization of Palm Oil Midrib Biochar as Soil Amendment with a Newly Isolated Bacillus sp. SM11 for Growth Enhancement and Nitrate Reduction in Romaine Lettuce (Lactuca sativa L. var. longifolia)" Agriculture 13, no. 10: 1986. https://doi.org/10.3390/agriculture13101986

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