Effects of biochar and biofertilizer on cadmium-contaminated cotton growth and the antioxidative defense system

Heavy metal pollution of agricultural soils poses significant risks to crop safety and environmental health. Soil heavy metals originate from parent materials and from anthropogenic inputs such as excessive pesticides, fertilizers, wastewater, and sewage sludge. Cadmium (Cd) is especially problematic due to its mobility, low threshold concentration for toxicity, persistence, and carcinogenicity. Even at relatively low accumulation levels in cotton compared to other metals, Cd can severely disrupt plant physiology, respiration, and growth. Previous studies report that excessive Cd leads to chlorosis, wilting, metabolic disorders, and declines in photosynthetic system performance and rate, ultimately reducing yield. Various amendments, including metal oxides, rock phosphate, polymers, organic compost, biochar, and microorganisms, have been explored to mitigate Cd toxicity. Biochar is an environmentally friendly amendment with high carbon content, large surface area, functional groups, and negative charge, shown to improve chlorophyll, photosynthesis, transpiration, stomatal conductance, and antioxidant enzyme activities. Microbial inoculants, notably Bacillus sp., can chelate and adsorb heavy metals, supporting photosynthesis and crop quality. In China, cotton is a key cash crop, predominantly in Xinjiang, where long-term agrochemical use has enriched Cd, Pb, Cu, and Zn in soils and cotton organs. Using cotton straw biochar can reduce secondary pollution, and biofertilizer can enhance yield, soil fertility, and pest resistance. This study therefore tested biochar and biofertilizer, alone, under graded Cd stress, to: (1) evaluate effects on Cd accumulation in cotton organs; (2) examine impacts on oxidative stress responses, photosynthesis, growth, and development; and (3) clarify how enhancing the antioxidant defense system can alleviate Cd’s negative effects on chlorophyll synthesis, photosynthesis, and growth.

Prior research shows that Cd accumulation in plants induces chlorosis, wilting, metabolic disturbances, and decreases in photosynthetic capacity and rate, leading to yield losses. Specific reports indicate notable heavy metal accumulation in cotton organs and substantial reductions in chlorophyll and photosynthesis even at low Cd exposures. Amendments such as biochar have improved plant physiological performance by increasing chlorophyll content, photosynthesis, transpiration, stomatal conductance, and antioxidant enzyme activities (POD, CAT), while reducing metal bioavailability. Microbial inoculants (e.g., Bacillus spp.) possess effective metal-chelating functional groups and can biosorb heavy metals, improving crop photosynthesis and quality. In Chinese cotton systems, heavy metal enrichment from agrochemical use has been documented in soils and plant tissues. Combining biochar (for immobilization via surface complexation, precipitation, ion exchange) with biofertilizers (biosorption and plant growth promotion) is a promising approach to mitigate Cd uptake and toxicity.

Soil collection and properties: Agricultural field soil was collected, residues removed, air-dried, and sieved (5 mm). Baseline properties included pH 7.76, organic matter 14.73 g kg⁻¹, total N 0.46 g kg⁻¹, total P 28.42 g kg⁻¹, total K 246.83 g kg⁻¹, and total Cd 0.25 mg kg⁻¹ (analytical methods: pH in 1:2.5 soil:water; conductivity at 25 °C; standard methods for organic matter, N, P, K; Cd by aqua regia digestion and atomic absorption). Biochar: Cotton straw biochar was produced per prior methods, air-dried, sieved (5 mm), and characterized (pH 9.50, organic matter 625 g kg⁻¹, total N 0.89 g kg⁻¹, total P 2.54 g kg⁻¹, total K 8.62 g kg⁻¹, total Cd 0.002 mg kg⁻¹; functional groups measured: carboxyl 0.20 mmol g⁻¹, lactone 0.25 mmol g⁻¹, phenolic hydroxyl 0.21 mmol g⁻¹). Biofertilizer: Commercial Bacillus composite (≥20×10⁹ CFU g⁻¹; >99.6% Bacillus; moisture <10%; pH 7.8; total Cd 0.0001 mg kg⁻¹; per Chinese standard GB 20287-2006). Preparation of contaminated soil: CdCl₂·2.5H₂O was dissolved to 1.2 g L⁻¹ Cd²⁺ stock. Aliquots (10, 20, 40 mL) were mixed with 12 kg soil to achieve 1, 2, 4 mg Cd kg⁻¹; 0 mg kg⁻¹ served as control. Contaminated soils were equilibrated for 60 days. Experimental design: Completely randomized design with 12 treatments and five replicates each in pots (25 cm × 40 cm). Treatments: four Cd levels (0, 1, 2, 4 mg kg⁻¹) crossed with: no amendment (B0/J0), 3% w/w biochar (B1), or 1.5% w/w biofertilizer (J1). Treatment codes: H0B0, H0B1, H0J1, H1B0, H1B1, H1J1, H2B0, H2B1, H2J1, H3B0, H3B1, H3J1. Pots were maintained at 60% field capacity with deionized water. Basal fertilization per pot equivalent to N-P₂O₅-K₂O 180-150-210 kg ha⁻¹ (urea, diammonium phosphate, potassium sulfate): all P and K and half N pre-sowing; remainder post-planting. Plant material and growth: Cotton (Gossypium hirsutum L., cv. Xinluzao 53) seeds were surface-sterilized (2.5% NaOCl). Twenty seeds were sown per pot; at 3 true leaves, five uniform seedlings per pot were retained. Irrigation used deionized water. Samples (roots, stems, leaves, bolls) were harvested at boll stage, washed, fresh weights recorded, then oven-dried (105 °C 2 h kill, then 85 °C to constant weight) for dry weight. Cd analysis and transfer coefficients: Dried tissues (0.5 g) were digested (HNO₃:HClO₄ = 2:1) under pressure/temperature; Cd quantified by atomic absorption spectrophotometry. Transfer coefficients defined as F1 = stem Cd/root Cd; F2 = leaf Cd/stem Cd; F3 = boll Cd/stem Cd. Antioxidant enzymes and stress markers: SOD activity by photochemical NBT reduction; CAT and POD activities per Cakmak and Marschner; malondialdehyde (MDA) via TBARS; electrolyte leakage (EL) per standard method. Photosynthetic pigments and gas exchange: Chlorophyll a, b and carotenoids extracted with 80% acetone and quantified at 663.2, 646.5, 470 nm using Lichtenthaler equations. Gas exchange parameters (net photosynthetic rate Pn, stomatal conductance Gs, intercellular CO₂ concentration Ci, transpiration rate Tr) measured with LI-6400. Statistics: Data compiled in Excel; regression tests and multiple comparisons (Duncan’s new multiple range test, α=0.05) in SPSS 23.0. Redundancy analysis (RDA) performed to relate growth indices and physiological variables; graphs in Origin 8.0.

- Biomass and growth: Exogenous Cd significantly reduced cotton total dry weight (by ~16–17% at 1–2 mg kg⁻¹; lowest total dry weight 60.63 g at 4 mg kg⁻¹). Amendments rescued growth. Relative to H0B0, biochar (H0B1) increased root, stem, leaf, and boll dry weights by 37.96%, 0.58%, 43.27%, and 13.24%, respectively; biofertilizer (H0J1) increased roots 58.01%, stems 3.22%, bolls 43.64%, leaves 40.11%. Maximum total dry weight reached 91.17 g (H0J1). Across Cd levels (1–4 mg kg⁻¹), similar positive trends were observed. - Cd accumulation and transport: Roots were the main Cd sink (max 0.291 mg kg⁻¹), followed by leaves, stems, and bolls. Increasing exogenous Cd increased Cd in all organs. Compared with H0B0, H2B0 and H3B0 increased Cd in roots by 17.39% and 40.58%, leaves by 11.32% and 35.85%, stems by 28.86% and 32.21%, and bolls by 15.53% and 31.07% (P<0.05). Amendments significantly reduced Cd accumulation, especially in bolls (P<0.05). Under H3, biochar (H3B1) and biofertilizer (H3J1) reduced Cd in roots by 20.27% and 17.87%, leaves by 4.63% and 11.11%, stems by 15.23% and 15.74%, and bolls by 26.67% and 8.89%, respectively, relative to H3B0. Transfer coefficients: F2 (stem→leaf) > F1 (root→stem) and F3 (stem→boll). Biochar and biofertilizer significantly reduced F1 (e.g., H2B0 F1=0.079 reduced to 0.058 with H2B1 and 0.061 with H2J1), while increasing stem→leaf transfer. - Photosynthetic pigments: Exogenous Cd reduced chlorophyll a, b, and carotenoids (e.g., at H3B0 vs H0B0: Chl a −22.43%, Chl b −29.73%, Car −7.25%). Amendments increased Chl a and Chl b (P<0.05). At H3, biochar and biofertilizer raised Chl a by 42.98% and 69.84% and Chl b by 31.92% and 37.55%, respectively, vs H3B0. Carotenoids increased notably under amendments; at H0, Car rose 18.44% (H0B1) and 31.56% (H0J1) vs H0B0. - Gas exchange: Cd significantly decreased Pn, Gs, Ci, and Tr (max reductions vs H0B0 up to 67.76%, 88.89%, 43.83%, and 78.71%, respectively). Amendments increased these parameters. Relative to no amendment, biochar yielded maximum increases of Pn 26.11%, Gs 268.7%, Ci 92.65%, Tr 203.6%; biofertilizer yielded maximum increases of Pn 112.60%, Gs 32.92%, Ci 92.65%, Tr 128.20% (depending on Cd level and treatment). - Antioxidant system and stress markers: Cd decreased SOD and CAT activities in leaves and roots and increased MDA and EL. Amendments significantly increased SOD and CAT (leaves) and CAT (roots), while reducing MDA and EL (P<0.05). Leaf SOD and CAT increases with biochar were up to 68.97% and 40.80% (H0B1). With biofertilizer, leaf SOD and CAT rose up to 113.9% and 70.29% (H3J1). Root SOD increases reached 117.6% (H3B1) and 119.8% (H3J1). Cd-induced maxima: leaf and root MDA of 25.045 and 9.994 µmol mg⁻¹ and highest EL under H3B0. Amendments reduced leaf MDA and EL by up to 13.39% and 29.34% (biochar; H1B1 and H3B1) and by 14.42% and 13.98% (biofertilizer; H2J1 and H1J1) compared with respective no-amendment Cd treatments. - Multivariate relationships (RDA): First two axes explained 72.68% of variance (RDA1 52.56%, RDA2 20.12%). Cd contents in leaves, stems, roots were positively associated with POD, EL, and MDA and negatively associated with growth indices (dry weight), chlorophyll, photosynthesis, and SOD/CAT. Treatments without amendments under Cd (H1B0, H2B0, H3B0) clustered with higher Cd, MDA, and EL; amended treatments aligned with improved photosynthesis, pigments, and antioxidant enzyme activities.

The findings confirm that Cd is highly detrimental to cotton physiology and growth, depressing chlorophyll content, photosynthetic performance, and antioxidant enzyme activities while increasing oxidative damage (MDA) and membrane injury (electrolyte leakage). Biochar and biofertilizer effectively mitigated these impacts by reducing Cd uptake and translocation (notably root→stem), thereby lowering Cd burdens in stems and bolls, and by enhancing antioxidant defenses (SOD, CAT) that detoxify reactive oxygen species. Mechanistically, biochar likely immobilizes Cd via surface complexation, precipitation, and ion exchange, whereas Bacillus-based biofertilizer contributes through biosorption and cellular sequestration, reducing bioavailable Cd and supporting plant physiological functions. Improved pigments and gas exchange under amendments indicate alleviation of Cd-induced photosynthetic inhibition. RDA corroborated these relationships, showing negative correlations between tissue Cd and key growth/physiological indices and positive correlations with stress markers. Collectively, the amendments address the research aims by decreasing Cd accumulation in organs (especially bolls), enhancing antioxidant defense, and restoring photosynthesis and growth under Cd stress.

Biochar (3%) and Bacillus-based biofertilizer (1.5%) effectively reduced Cd accumulation in cotton organs and significantly decreased Cd migration from roots to stems (P<0.05), strongly inhibiting Cd transfer to stems and bolls. Across Cd levels, stem Cd decreased on average by 27.50% (biochar) and 25.14% (biofertilizer). Exogenous Cd suppressed chlorophyll synthesis, photosynthesis, and antioxidant enzyme activities, impairing growth, whereas both amendments counteracted these effects by enhancing SOD and CAT activities, lowering MDA and electrolyte leakage, and improving chlorophyll content and gas exchange, ultimately promoting biomass accumulation. These results support the use of biochar and biofertilizer as effective soil amendments for mitigating Cd toxicity and improving cotton performance in contaminated soils. Future research could explore combined applications, field-scale trials, long-term effects on soil health and Cd dynamics, and optimization of amendment rates and microbial consortia.