Exploring Shrimp-Derived Chitin Nanofiber as a Sustainable Alternative to Urea for Rice (Oryza sativa cv. BRRI dhan67) Cultivation

Abstract

Rice is a staple food for nearly half the world population. Rice cultivation relies heavily on urea fertilization. However, the use of urea is prone to significant losses and contributes to environmental pollution. This study was aimed at fabricating nitrogen-rich chitin nanomaterials and assessing their effects on the growth and yield of rice. Chitin nanofibers (ChNF), with widths ranging from 10 to 30 nm, were successfully isolated from shrimp shells by chemical pretreatment and mechanical fibrillation. Pot-grown rice plants were treated with various concentrations of ChNF and urea in a completely randomized design with five replicates. ChNF treatment resulted in plant height (97.33 ± 1.53 cm), tiller number (17.67 ± 1.15 hill⁻¹), straw yield (30.40 ± 1.93 g hill⁻¹), and harvest indexes comparable to those achieved with urea treatment at harvest (97.33 ± 1.53 cm, 17.00 ± 1.73 hill⁻¹, 26.47 ± 2.39 g hill⁻¹ and 44.12%, respectively). The grain yield using urea (22.70 g hill⁻¹) was almost identical to that achieved with 0.01% ChNF (22.22 g hill⁻¹), which may be attributable to the increased nitrate-nitrogen (N) and ammonium-N availability, reduced nitrogen loss, and enhanced microbial activity associated with 0.01% ChNF. The study findings indicate that shrimp-derived ChNF is a promising functional nanomaterial for rice cultivation, with potential as a partial or full replacement for urea in sustainable rice production.

1. Introduction

Rice (Oryza sativa L.) is a crucial crop in developing countries and a staple for approximately half the world population. The consumption of rice increased from 437.18 million metric tons in 2008–2009 to 520.4 million metric tons in 2022–2023 [1]. As the global population increases, the demand for rice continues to increase and is expected to reach 584 million tons by 2050. Efforts have been made to enhance rice production using high-yield varieties, appropriate inputs, and management practices.

The use of chemical fertilizers significantly increases rice production. Urea, a nitrogenous fertilizer, has been used extensively and has greatly contributed to higher rice yields. However, the nitrogen use efficiency (NUE) of urea fertilizer is very low (30–35%) and the recovery of nitrogen from wetland rice seldom exceeds 40% [1]. Substantial amounts are lost through denitrification, volatilization, leaching, and runoff, leading to environmental pollution. Approximately 26% of the nitrogen in broadcast urea is lost as gaseous nitrogen through the nitrification–denitrification process and NH₃ volatilization [2]. After application to the soil, urea is hydrolyzed in the oxidized layer, forming NO₃⁻, which leaches from the soil and contaminates groundwater.

Recent research has focused on identifying organic substitutes that can replace urea without sacrificing high yields. Chitin, a linear polymer consisting of (1–4)-linked 2-acetamido-2-deoxy-β-d-glucopyranose, is biodegradable, nontoxic, and biocompatible. Among the various applications of chitin, its potential use as a soil fertilizer or amendment to enhance the growth of crops is important. Chitin contains high levels of nitrogen and has a very low carbon-to-nitrogen ratio [3], making it a valuable nitrogen-based organic fertilizer. However, the practical application of chitin is limited by its insolubility in most common solvents, which is attributed to its highly extended hydrogen-bonded crystalline structure. Chitin becomes water soluble only when it is heavily degraded by chemical modifications, a process that is not cost-effective. Therefore, chitin derivatives, such as chitosan or oligosaccharides, which are soluble in acidic water and other common solvents, are used in agricultural applications. However, these water-soluble chitin derivatives are expensive and are primarily used in experimental trials.

Chitin also plays a role as an inducer of plant defense against diseases [4]. These biopolymers, along with their deacetylated derivatives, have various unique functions that can improve crop yield. Nanofibrillated chitin functions as an antifungal, antibacterial, and antiviral agent [5]. Studies have shown a decrease in the proportion of empty grains, an increase in the number of tillers per plant, and an increase in dry matter buildup [6]. Chitin nanofibers promoted plant growth by increasing nitrogen uptake [7]. Chitosan solutions in acidic water have been shown to boost rice yield by approximately 31% [8]. Chitin nanofibers (ChNF) possess a large surface area, an intricate nanofiber network, remarkable biological properties, a low C/N ratio, and high nitrogen content, making them a promising eco-friendly fertilizer. Their unique morphological characteristics and high water solubility may enhance symbiotic plant-microbial interactions, improving nutrient availability and stimulating plant growth. To the best of our knowledge, no study has yet investigated the effects of chitin nanofibers on rice growth and yield. Therefore, this study aims to fabricate water-soluble ChNF from shrimp waste and evaluate its impact on rice growth and yield. A pot experiment was conducted using a completely randomized design with five replications. The findings confirm that shrimp-based ChNF is a promising functional nanomaterial for enhancing rice growth and yield, with the potential to partially or fully replace urea as a nitrogen source. This information could be valuable for addressing environmental concerns and minimizing the use of chemical fertilizers in Bangladesh and other parts of the world.

2. Materials and Methods

2.1. Preparation of Chitin Nanofibers from Shrimp Shell

Black tiger (Peneus monodon) shrimp shells were obtained from the shrimp-processing industry (Khulna, Bangladesh) and crushed into smaller particles (2–4 mm) using a grinder. Matrix substances in the shrimp shells were removed using a conventional method (Figure 1) [9]. The shells were treated twice with 7% HCl for 24 h at ambient temperature to completely remove the minerals. To remove the proteins completely, the demineralized shells were dipped in 4% NaOH solution in a beaker and heated at 80 °C for 6 h, and the procedure was repeated one more time. A slurry (1 wt.%) was prepared and subjected to mixing with a high-speed blender (Vita-Mix Blender, Entrex, Ltd., Tokyo, Japan) at 37,000 rpm for 10 min. The chitin suspension was then passed through a multifunctional mill, a Super Masscolloider (MKCA6-5J, Masuko Sangyo, Saitama-ken, Japan), for 10 cycles. The clearance of the grinding stone in the mill was adjusted to −0.15 mm from the zero position (slight contact) of the two stones and the rotating speed of the stone was set to 1500 rpm. The dispersion was collected and stored in a refrigerator.

2.2. Characterization of Chitin Nanofibers from Shrimp Shell

The chitin nanofiber suspension was subjected to oven-drying at 105 °C, after replacement of water with ethanol, and the obtained sheets were coated with an approximately 2 nm layer of platinum using an ion sputter coater and observed under a field emission scanning electron microscope (SEM, JSM-6700F, JEOL Ltd., Tokyo, Japan).

The Fourier transform infrared (FT-IR) spectra of shrimp shells, shrimp shell chitin, and crab shell chitin were obtained using an FT-IR spectrophotometer equipped with an ATR (Spectrum 65, Perkin-Elmer Japan Ltd., Yokohama, Japan). The measurement was performed over a wavenumber range of 550 to 4000 cm⁻¹ with 32 scans at a resolution of 4 cm⁻¹.

The chitin nanofibers’ crystalline structure was examined using X-ray diffraction (XRD). An X-ray generator (Ultima IV, Rigaku, Tokyo, Japan) operated at 40 kV within a goniometer scanning range of 5–35° was used to obtain the data.

2.3. Plant Materials and Cultivation Conditions

The pot experiment was conducted in an open location at Khulna University, geographically extended between 22°48′16.74″ N and 89°32′17.232″ E, Bangladesh. The climate at the research site was characterized by relatively high temperatures, substantial rainfall, and frequent strong winds during the Kharif season (April–September), and low temperatures and low humidity during the Rabi season (October–March). A high-yielding Boro rice variety, BRRI (Bangladesh Rice Research Institute) dhan67, was used as the planting material. Two-hundred kilograms of soil was mixed with various fertilizers: TSP (9.73 g), MoP (11.98 g), gypsum (9.73 g), and zinc sulfate (1.12 g), as per the BRRI recommendations. Thereafter, 10 kg of soil was placed in a 20 kg red plastic pot with the help of a spade.

The experiment was set up in a completely randomized design (CRD) using five treatments and five replicates. The following treatments were used: T1 = control (water); T2 = urea (recommended dose); T3 (ChNF only) = 0.01%; T4 (ChNF only) = 0.05%; and T5 (ChNF only) = 0.1%. Urea and ChNF were applied in three splits. The ChNF was carefully applied to allow the soil absorption and facilitate absorption by the plants. The first dose of ChNF and urea was applied at 18 days after transplanting (DAT). The second dose was applied at the active tillering stage (35 DAT) and the third dose was applied at the panicle initiation stage (60 DAT).

2.4. Measurements of Plant Growth Parameters and Inorganic Elements

To determine the different growth parameters, data were recorded, starting 13 DAT, and continuing until maturity. Plant height was measured from the base to the tip of the tallest leaf or panicle of each plant in each pot five times at 13, 20, 27, 34, 41, 48, and 55 DAT and at harvest. The number of tillers per hill was recorded and the means were computed on the same dates. The chlorophyll content of a single mature leaf of each plant was also recorded using a SPAD 502 Plus Chlorophyll meter (Konica Minolta Japan, Inc., Tokyo, Japan) on these dates [10].

The crop was harvested at full maturity, when 90% of the grains turned golden yellow. The plants from each pot were uprooted, bundled separately, and tagged appropriately. Grain and straw yields were also recorded. The plants were then dried under the sun. After separating the grains from the straw, they were weighed. After harvesting, the number of effective tillers per hill was recorded, and the mean was calculated. The panicle length was measured for each effective panicle from the base node of the rachis to the apex of each panicle, and the mean was calculated. The number of grains per hill was counted, and the average was calculated. The total number of unfilled spikelets per panicle was also counted and the means were calculated. After sun drying, 1000 grains from each treatment were weighed on an electric balance to obtain the 1000-grain weight. Similarly, the grain weight was recorded separately and expressed as g hill⁻¹. The straw weight of the harvested crop from each pot was determined. A sample from each pot was dried in an oven at 60 °C for 72 h, and the weight (g hill⁻¹) was recorded separately pot wise. To determine the biological yield, the grain and straw yields were added and the ratio of grain yield to biological yield was expressed as the harvest index (%) [10].

The soil, rice grain, and straw samples were ground, dried in an oven at 50 °C for approximately 24 h, and stored in poly bags. Total N% was calculated by subjecting the fine particles of soil, rice straw, and rice grain to a Carbon and Nitrogen Elemental Analyzer (CN 802, Velp Scientifica, Usmate, Italy) at 1030 °C for 5 min.

2.5. Bacterial Count

A 1:100 (10⁻²) dilution of soil was prepared by weighing 1 g of soil and adding 99 mL sterile water. Fifteen milliliters of nutrient agar medium was poured into each appropriately labeled Petri plate. One milliliter of the diluted sample was added onto the surface of the nutrient agar in Petri plates from each serially diluted test tube. All the plates were incubated in an inverted position for approximately 24 h at 37 °C. After incubation, a plate with a countable number of colonies (containing 30–300 colonies) was selected. The number of colonies was counted using a colony counter and the number of colonies in 1 mL of the original sample was calculated as follows:

Colony forming unit (CFU)/mL = Colonies/(Amount plated × dilution)

2.6. Stastical Analysis

Data were analyzed using a one-way analysis of variance (ANOVA) with IBM SPSS Statistics 25. Treatment means were separated using Tukey’s HSD Test at 5% probability (p < 0.05).

3. Results and Discussion

3.1. Characteristics of Chitin Nanofibers from Shrimp Shells

Shrimp shells are composed of a complex polymer matrix containing varying amounts of protein, lipids, and minerals, which strengthen the α-chitin nanofibers. To extract chitin from this matrix, a series of decalcification and deproteinization steps using acid and alkali treatments were employed [9]. Despite these chemical processes, the shells retained their morphological structure.

Another approach involves a deproteinization process using enzymes, which can occur at low temperatures. However, achieving complete protein removal is challenging [11]. Additionally, a new method for extracting chitin employs a Deep Eutectic Solvent. While this method is environmentally friendly, it is not appropriate for ChNF production since it dissolves and breaks down chitin crystals [12].

After mechanical treatment, the shells became soft, viscous, and white.

The stability of ChNF was evaluated by observing its water dispersion, which remained homogeneous for more than two months without any precipitation. This strongly suggests that the ChNF was successfully developed through chemical treatments and mechanical fibrillation and formed a favorable fiber network in water.

To confirm the successful extraction of nanofibers from the shrimp shells, the morphology of the ChNF was investigated using field emission scanning electron microscopy (FE-SEM). Figure 2 shows SEM images of raw chitin and ChNF at different magnifications. Figure 2a shows the structure of the chitin particles with intact fibers visible. In contrast, Figure 2b,c show an absence of massive particles and a significant decrease in fiber diameter, resulting in a vast network of entangled fibers. This indicated that mechanical fibrillation effectively reduced the diameter of the chitin fibers and promoted fiber-to-fiber interactions. This morphology was similar to that of ChNF obtained from crab shells via mechanical grinding [9]. The diameter distribution of the nanofibers fibrillated at 10 passes, evaluated using SEM micrographs, was 10–30 nm for approximately 80% of the nanofiber. The value is nearly identical, as the chitin nanofibers sourced from crab shells processed with the same grinder measure 10–20 nm [9].

The FT-IR spectrum of the shrimp shell (Figure 3, black line) revealed a broad peak at 3276 cm⁻¹, which was attributed to the O-H and N-H stretching vibrations. Additionally, the small peak at 2920 cm⁻¹ corresponded to C-H stretching vibrations (CH₃ and CH₂). Signals at 1635 and 1530 cm⁻¹ corresponded to amide I (C=O stretching) and amide II (N-H bending) bands, respectively, indicative of proteins, and are observed in the FT-IR spectrum of chitin owing to its C=O and N-H groups. Bands at 1780 and 870 cm⁻¹ indicated the stretching and bending vibrations of CaCO₃. Peaks at 1450 and 1380 cm⁻¹ signified CH₃ bending, whereas the signal at 1025 cm⁻¹ corresponded to C-O-C stretching vibrations [13,14]. Figure 3 (red line) shows the FT-IR spectrum of chemically extracted shrimp chitin. The functional groups, vibration modes, and wavenumbers were listed in

Table 1. Fourier transform infrared spectrum data of chitin.

Functional Group/Vibration Mode	Wavenumber (cm−1)
O–H/stretching	3435
N–H/stretching	3255
N–H/stretching	3100
CH3/stretching	2880
C=O/Amide I	1620
N–H/Amide II	1550
CH/bend, CH3/symmetry	1380
CH3/wagging	1305
C–O/asymmetry, stretch	1005
CH/ring stretching	870

. Signals at 3100–3435 cm⁻¹ were attributed to O-H and N-H stretching vibrations, whereas C-H stretching vibrations appeared at 2880 cm⁻¹. The characteristic signals of shrimp chitin included peaks at 1620 and 1550 cm⁻¹, corresponding to the stretching vibrations of amide I and amide II (C-N stretching), respectively. A doublet signal (1650 and 1620 cm⁻¹) for amide I indicated the α-allomorph of chitin, as its β-form appeared as a single band. Peaks at 1425 and 1380 cm⁻¹ corresponded to CH₃ bending. The signal at 1005 cm⁻¹ was attributable to the C-O-C bond. The absence of peaks at approximately 1780 and 870 cm⁻¹ indicated the complete removal of CaCO₃ [13,14,15]. The spectrum of shrimp chitin was in excellent agreement with that of crab chitin (Figure 3, blue line).

Figure 4 illustrates the X-ray scattering patterns of chitin extracted from shrimp shells. The prominent diffraction peaks indicate that this chitin has a crystalline structure, which is consistent with findings reported in the literature for chitin obtained from crab shells. Specifically, the reflection peaks observed at 9.2° and 19.1° correlate with the (020) and (110) diffraction planes of the orthorhombic system, respectively [16].

3.2. Effects of ChNF on the Growth Attributes and Yield of Rice

The application of varying concentrations of ChNF throughout the growing period positively influenced plant height compared with that of the control (Figure 5). Although no significant differences in plant height were observed among the treatments at any of the growth stages, the tallest plants (97.33 ± 1.53 cm) were recorded in the urea- and ChNF-treated groups at harvest, whereas the shortest plants were found in the control treatment (88.33 ± 6.66 cm) (Figure 5).

Similarly, the number of tillers per hill showed no significant differences during the early growth stages (13, 20, 27, and 34 DAT). However, at later stages (41, 48, and 55 DAT, as well as at harvest), the number of tillers was significantly higher in the ChNF and urea treatments than in the control treatment (

Table 2. Effects of ChNF on tiller number hill⁻¹ for BRRI dhan67 at different days after transplanting (DAT).

Tiller Number Hill−1
Treatment	13 DAT	20 DAT	27 DAT	34 DAT	41 DAT	48 DAT	55 DAT	At Harvest
Control	17.75 ± 2.63	16.50 ± 1.29	15.75 ± 1.89	15.33 ± 1.53	12.33 ± 0.58 b	11.33 ± 0.58 b	11.00 ± 1.00 b	11.00 ± 1.00 b
Urea	18.25 ± 5.31	20.00 ± 2.71	19.75 ± 2.06	21.00 ± 3.00	19.67 ± 2.52 a	19.00 ± 1.73 a	17.67 ± 1.53 a	17.00 ± 1.73 a
0.01% ChNF	22.75 ± 2.50	19.75 ± 2.06	19.25 ± 2.75	18.67 ± 3.05	17.67 ± 1.53 a	17.00 ± 1.73 a	16.33 ± 1.15 a	16.33 ± 1.15 a
0.05% ChNF	21.75 ± 1.71	19.75 ± 2.36	17.75 ± 0.50	17.67 ± 1.53	16.67 ± 0.58 a	16.67 ± 1.53 a	15.67 ± 1.15 a	15.33 ± 1.53 a
0.1% ChNF	20.00 ± 3.83	19.00 ± 1.41	19.25 ± 1.89	19.00 ± 2.65	19.33 ± 0.58 a	18.00 ± 1.00 a	17.67 ± 1.15 a	17.67 ± 1.15 a

Data represent mean ± SD (standard deviation) of five replicates. In a column, means followed by similar letter(s) are not statistically different according to Tukey’s HSD Test (p < 0.05).

). The number of tillers in the urea and all ChNF treatments were statistically similar, but were significantly greater than that in the control. At harvest, the highest tiller number per hill (17.67) was observed in the 0.1% ChNF treatment, which was comparable to that of urea (17.00), and with 0.01% ChNF (16.33) (Table 2).

There were significant differences in the leaf chlorophyll content index among the treatments at all growth stages, except at harvest (

Table 3. Effects of ChNF on the leaf chlorophyll content index of BRRI dhan67 on different days after transplanting (DAT).

Leaf Chlorophyll Content Index
Treatment	13 DAT	20 DAT	27 DAT	34 DAT	41 DAT	48 DAT	55 DAT	At Harvest
Control	37.28 ± 1.65 bc	33.50 ± 2.02 b	32.65 ± 0.83 b	33.17 ± 2.76 ab	34.33 ± 2.73 b	29.63 ± 2.25 b	31.87 ± 2.25 b	31.97 ± 4.35
Urea	40.95 ± 0.98 a	35.78 ± 1.11 ab	37.03 ± 0.60 a	37.83 ± 1.50 ab	37.07 ± 1.51 ab	39.53 ± 2.17 a	36.33 ± 3.07 ab	36.70 ± 3.76
0.01% ChNF	39.65 ± 0.51 ab	32.30 ± 1.47 b	33.38 ± 2.46 b	33.03 ± 1.17 ab	35.63 ± 1.57 ab	33.73 ± 1.33 ab	33.00 ± 0.56 b	32.90 ± 5.53
0.05% ChNF	36.78 ± 0.94 c	34.38 ± 1.49 ab	32.48 ± 1.49 b	32.60 ± 1.51 b	35.33 ± 2.06 ab	35.80 ± 3.46 ab	34.90 ± 2.33 ab	35.83 ± 3.05
0.1% ChNF	37.08 ± 1.43 c	37.53 ± 1.96 a	37.35 ± 0.52 a	38.03 ± 2.76 a	39.93 ± 1.95 a	39.20 ± 2.42 a	40.13 ± 2.91 a	39.33 ± 4.73
p	0.001	0.004	0.001	0.014	0.049	0.003	0.014	0.305

Data represent mean ± SD (standard deviation) of five replicates. In a column, means followed by similar letter(s) are not statistically different according to Tukey’s HSD Test (p < 0.05).

). In most cases, the ChNF treatments and urea had similar chlorophyll content, whereas the control had the lowest values. At 55 DAT, the highest chlorophyll index (40.13) was obtained with 0.1% ChNF, which was comparable to that for urea treatment (36.33), whereas the control treatment had the lowest value (31.87). Chitin nanofibers promoted the growth of komatsuna and tomato plants [17], and their application increased plant biomass and leaf chlorophyll content in tomatoes [7]. Chitosan, alone or in combination with chemical fertilizers, promoted rice growth without affecting plant height [18,19,20,21,22]. Chitosan powder and oligomers increased the growth of rice (BRRI dhan29) and coffee seedlings [22].

Although ChNF increased the tiller number and leaf chlorophyll content index (SPAD value) in rice compared with that in the control, it had no effect on plant height. Several studies have reported that chitosan increases the number of tillers and the leaf chlorophyll index in rice [23] and wheat [24]. A decrease in the quantity of empty grains was noted, but an increase in the number of tillers per plant and a build-up of dry matter was observed [4]. ChNF application was reported to promote plant growth by increasing the nitrogen uptake [7]. However, treatment with 0.01% ChNF and the recommended dose of urea resulted in statistically similar plant heights, tiller numbers, leaf chlorophyll content, and plant biomass production, indicating that ChNF has the potential to enhance rice growth and physiological attributes.

All yield attributes and rice yield, except the 1000-grain weight and harvest index, were significantly affected by ChNF application. The highest number of panicles per hill was obtained with 0.1% ChNF application (18), which was significantly different from that for the control (11.67) and similar to that for urea (17.67) and 0.01% ChNF (17.67) (

Table 4. Effects of ChNF on yield attributes and yield of BRRI dhan67.

Treatment	Effective No. of Panicle Hill−1	Panicle Length (cm)	Filled Grain (No.) Hill−1	Unfilled Spikelet (No.) Hill−1	Total Grain (No.) Hill−1	1000-Grain Weight (g)	Grain Yield (g)	Unfilled Spikelet Weight Hill−1 (g)	Straw Yield Hill−1 (g)	Biological Yield (g)	Harvest Index (%)
Control	11.67 ± 0.58 b	18.51 ± 0.33 b	722 ± 59.18 b	262 ± 144.36 b	984 ± 88.93 b	18.95 ± 0.91	13.70 ± 1.72 b	0.91 ± 0.56 b	16.45 ± 1.39 b	31.07 ± 1.93 b	44.11
Urea	17.67 ± 3.51 a	21.58 ± 0.40 a	1274 ± 157.29 a	562 ± 65.79 a	1836 ± 206.48 a	17.81 ± 0.80	22.70 ± 3.11 a	2.28 ± 0.66 a	26.47 ± 2.39 a	51.46 ± 4.72 a	44.12
0.01% ChNF	17.67 ± 1.53 a	20.71 ± 0.80 ab	1283 ± 142.32 a	532 ± 50.48 a	1815 ± 225.25 a	17.38 ± 0.77	22.22 ± 1.55 a	1.77 ± 0.18 ab	26.87 ± 1.21 a	50.86 ± 1.96 a	43.68
0.05% ChNF	16.33 ± 1.15 ab	20.46 ± 0.51 ab	1065 ± 158.03 ab	451 ± 47.88 ab	1516 ± 125.60 a	18.87 ± 1.12	20.01 ± 2.44 a	1.71 ± 0.24 ab	25.78 ± 2.29 a	47.49 ± 4.36 a	42.13
0.1% ChNF	18.00 ± 1.73 a	19.93 ± 1.99 ab	1197 ± 120.462 a	550 ± 90.05 a	1747 ± 203.34 a	17.55 ± 0.23	21.02 ± 2.25 a	1.69 ± 0.22 ab	30.40 ± 1.93 a	53.11 ± 4.17 a	39.58
p	0.014	0.041	0.002	0.009	0.001	0.115	0.004	0.033	0.001	0.001	0.67

Data represent mean ± SD (standard deviation) of five replicates. In a column, means followed by similar letter(s) are not statistically different according to Tukey’s HSD Test (p < 0.05).

). The longest panicle length was recorded for urea (21.58 cm), which was statistically similar to that for 0.01% ChNF (20.71 cm). The highest number of filled grains per hill was observed with 0.01% ChNF (1282.33), whereas the control had the lowest number (721.67). The highest number of unfilled spikelets per hill was obtained for urea (562), and the highest total number of grains per hill was recorded for urea (1836), which was statistically similar to that for all the ChNF treatments.

The highest grain yield per hill was achieved for urea (22.70 g), which was statistically similar to that for 0.01% ChNF (22.22 g), 0.05% ChNF (20.01 g), and 0.1% ChNF (21.02 g) but significantly different from that for the control (13.70 g). Similarly, all ChNF treatments resulted in similar dry weights of unfilled spikelets, comparable to that for urea but significantly higher than that for the control treatment. Statistically similar straw and biological yields were recorded for urea and all ChNF treatments, although higher straw and biological yields were obtained with the highest ChNF concentration (0.1%). The harvest index was not significantly different among the treatments, with 0.01% ChNF (44.12%) resulting in a harvest index similar to that of urea (44.12%) (Table 4). Weather was favorable for rice cultivation during the growing period (Figure 6).

The application of ChNF and the recommended dose of urea significantly enhanced the key yield parameters, including the number of panicles, panicle length, filled grains, total grains, grain yield, straw yield, and overall biological yield, compared with the control treatment. Notably, the use of 0.01% ChNF resulted in a yield and yield attributes statistically comparable to those obtained with urea. These included the number and length of panicles, number of filled and total grains, grain and straw yields, biological yield, 1000-grain weight, and harvest index.

ChNF was reported to improve the NUE and biomass production in tomatoes, potentially by modulating the expression of genes associated with nitrogen uptake, nutrient allocation, and photosynthesis [7,17]. Additionally, it was reported to possess antifungal properties and suppressed Fusarium wilt in tomatoes [25]. Research on modified chitosan has also reported an increased tiller number, grain yield, straw yield, and biological yield in rice cultivar BRRI dhan62 [22], whereas chitosan solutions boosted rice yields by approximately 31% [26]. Given that 0.01% ChNF resulted in statistically similar yields and yield attributes to the recommended dose of urea, ChNF is a promising alternative to conventional urea fertilization for rice cultivation in Bangladesh.

To evaluate the effect of ChNF application on plant growth and yield, we analyzed the nitrogen content in both soil and plants, as well as treatment-specific nitrogen loss. While urea contains 46% nitrogen, ChNF has a significantly lower nitrogen content (3.6–6.8%) [27]. Despite this, the 0.01% ChNF treatment showed no significant differences compared with urea in terms of postharvest soil nitrogen, nitrate, and ammonium-N levels, as well as grain and straw nitrogen content. In all cases, ChNF exhibited numerically higher values, with 21% more nitrate-N and 5% more ammonium-N than urea. This suggested that 0.01% ChNF enhanced the availability of nitrate and ammonium nitrogen, potentially explaining its grain yield performance, which is comparable with that of urea (Figure 7).

Plants absorb nitrogen primarily in the form of nitrate (NO₃⁻) [28]. Urea fertilizers predominantly contain ammonium (NH₄⁺) ions, which must undergo conversion to nitrate before being utilized by plants—a lengthy process. In contrast, ChNF contains a higher proportion of nitrate ions than conventional urea fertilizers [17]. Upon application to soil, urea hydrolyzes in the oxidized layer, forming NO₃⁻. However, because nitrate ions are negatively charged, they are not retained in soil and are prone to leaching. Chitin nanofibers, in contrast, possess a unique hydrophobic structure with a high surface-to-volume ratio [29] allowing them to retain more water and nutrients, including nitrogen. This enables a slow and sustained release of nitrogen, ensuring its availability to plants over an extended period. However, increasing the ChNF concentration did not further enhance the nitrate uptake. At higher concentrations, ChNF may bind essential nutrients, reducing their availability for plant uptake, which could explain the lack of an additional increase in yield at higher ChNF doses (Table 4). This finding was further supported by the analysis of nitrogen uptake in both rice grains and straw per hill. The nitrogen uptake values at 0.01% ChNF were not significantly different from those observed with urea, indicating that ChNF application facilitated nitrogen uptake comparable to that achieved with conventional fertilization. Notably, the lowest concentration of ChNF yielded the most favorable results (

Table 5. Nitrogen (N) balance in soil and plants in rice cultivation with different concentrations of ChNF.

	Soil total N (%)
Initial soil	0.206 ± 0.006
Treatments	Initial soil N and added N (g)	Postharvest soil N (g)	Grain N (%)	Straw N (%)	Grain N uptake (g hill−1)	Straw N uptake (g hill−1)	Postharvest soil N and plant uptake N(g)	N lost (%)
Control	25.60	21.6 ± 0.004	1.283 ± 0.005	0.714 ± 0.028 ab	0.176 ± 0.01 b	0.11 ± 0.006b	21.89 ± 0.02 c	14.5 b
Urea	26.22	21.2 ± 0.003	1.275 ± 0.036	0.756 ± 0.011 a	0.291 ± 0.03 a	0.20 ± 0.01a	21.69 ±0.04d	17.3 a
0.01% ChNF	25.66	22.7 ± 0.005	1.291 ± 0.036	0.786 ± 0.019 a	0.286 ± 0.01 a	0.20 ± 0.006a	23.19 ± 0.01 a	9.6 d
0.05% ChNF	25.90	22.1 ± 0.006	1.230 ± 0.026	0.767 ± 0.013 a	0.247 ± 0.020 ab	0.18 ± 0.01a	22.43 ±0.03b	13.4 c
0.1% ChNF	26.12	22.0 ± 0.003	1.195 ± 0.012	0.663 ± 0.017 b	0.251 ± 0.01 ab	0.19 ± 0.006a	22.44 ±0.02b	14.1 b
p		0.146	0.112	0.0105	0.0064	0.0002	<0.0001	<0.0001

In a column, values followed by similar letters are not statistically different according to Tukey’s HSD Test (p ≤ 0.05). Values represent the averages of five replicates.

). Chitin-treated plants demonstrated higher NUE than untreated plants, contributing to improved NUE and enhanced nitrogen accumulation [7]. This, in turn, supports improved plant growth and stronger defense mechanisms [30]. Genetic modifications and the expression of genes related to nitrogen acquisition, assimilation, nutrient allocation, and photosynthesis may further enhance plant biomass, chlorophyll content, photosynthetic efficiency, and seed yield.

Soil microbes play a vital role in biochemical processes such as organic matter decomposition, detoxification of harmful substances, nitrogen fixation, and transformation of essential nutrients, such as nitrogen, phosphorus, and potassium. Our study revealed significant variations in the bacterial populations across different ChNF treatments. The highest bacterial population (88.1 × 10⁶ cfu/mL) was observed in the 0.01% ChNF treatment, which was statistically similar to that of urea (72.3 × 10⁶ cfu/mL). Notably, the lowest bacterial population (55.1 × 10⁵ cfu/mL) was recorded at the highest ChNF concentration (0.1%), which was comparable to that of the control treatment (

Table 6. Bacterial population count in soil treated with ChNF.

Treatments	cfu/mL
Control	73.4 × 105 ± 5.69 × 105 bc
Urea	72.3 × 106 ± 11.53 × 106 a
0.01% CNF	88.1 × 106 ± 10.60 × 106 a
0.05% CNF	82.9 × 105 ± 12.06 × 105 b
0.1% CNF	55.1 × 105 ± 8.74 × 105 c
p	<0.0001

Data represent mean ± SE (standard error) of three replicates. In a column, means followed by similar letter(s) are not statistically different according to Tukey’s HSD Test (p < 0.05).

).

The increased bacterial population in the 0.01% ChNF treatment may play a crucial role in enhancing nutrient availability for rice plants, thereby supporting higher growth and grain yield. Chitin nanofibers can act as substrates for beneficial soil microorganisms, including chitinolytic bacteria, which produce chitinases that break down chitin into simpler compounds and stimulate plant growth and yield [31]. The enzymatic degradation of chitin by endochitinases, exochitinases, and β-n-acetylhexosaminidases converts it into oligomers, which are further broken down into monomers that decompose into ammonia and nitrates, making nitrogen more readily available for plant uptake [32,33]. Thus, ChNF may create a favorable environment for specific bacterial populations, influencing their activity and distribution in the soil surrounding the roots of rice plant. When applied at low concentrations, ChNF can enhance microbial growth and activity, leading to improved soil fertility and nutrient cycling. Further research is required to fully understand the mechanisms by which ChNF promotes rice growth and yield.

Lizundia et al. calculated the pilot-scale production costs of chitin nanofibers [34]. The minimum selling price for chitin nanofibers derived from 3000 kg of mushrooms was EUR 212 per kg on a dry weight basis. While this is significantly higher than the cost of urea for fertilizers, chitin nanofibres could become competitive based on future trends in urea prices, particularly given their application as a low-concentration dispersion with lasting effects. Going forward, it will be important to confirm the optimal fertilization conditions for chitin nanofibers and assess their cost advantages compared to the current fertilizers.

4. Conclusions

In this study, chitin nanofibers (ChNF) were successfully fabricated from shrimp shells and their effects on rice growth and yield were evaluated. ChNF application significantly improved the growth and yield of the rice cultivar BRRI dhan67 compared with that in the control group. Notably, the 0.01% ChNF treatment resulted in plant growth, grain yield, straw yield, biological yield, and a harvest index comparable to those achieved with urea, highlighting its potential as a functional nanomaterial for sustainable agriculture. Partial or complete replacement of urea with ChNF could revolutionize global rice cultivation by enhancing nitrogen uptake while reducing environmental pollution. These findings hold significant promise for minimizing the use of chemical fertilizers, particularly in Bangladesh and other rice-producing regions worldwide. However, to establish definitive recommendations for ChNF application and optimal concentrations for rice grain yield, further field experiments across different regions of Bangladesh and multiple rice-growing seasons are necessary.