Collection and analysis of soil samples
The long term polluted top soils (<20 cm in depth) used for greenhouse experiments were sampled from an agricultural area in Pha Dei Village, Mae Sot District, Tak Province, Thailand (N 16° 40΄ 35.9˝ E 98° 37΄ 37.4˝) at an altitude of 197 m. The soil at this site was tilled for either rice-corn or rice-bean crops in one cropping year. The selected physicochemical characteristics of the soil are shown in Table 4-1 & 4-2. Soil samples were divided in two main portions: one for physicochemical characterizations and the other for enriched culture following biofertilizer preparation.
Soil material was homogenized, air-dried, crushed, and sieved (2-mm mesh size). The following physicochemical properties of the soil were determined: pH and electrical conductivity (EC) (1:5 soil/water suspensions) using a pH meter and an EC meter respectively; OM content by wet oxidization and titration according to the modified Walkley-Black procedure(Nelson &Sommers 1996) ; cation exchange capacity (CEC) using 1 N ammonium chloride pH 7.0 after pretreatment to remove soluble salts (Oorts et al. 2007); total N by the Kjeldahl method; extractable P by Bray II method (Bray &Kurtz 1945) and extractable K using an atomic absorption spectrophotometer (Perkin Elmer Analyst 200, USA) after ammonium acetate extraction at pH 7.
Preparation and analysis of Cd-resistant biofertilizer
The biofertilizers used as amendments for remediation of Cd contaminated soils were prepared using repeated culture enrichment of the soil Cd-resistant bacteria as previously described (Seang-On et al. 2019) followed by semi-solid fermentation/biofertilization conditions. Topsoil (<20 cm in depth) was collected from a long- term Cd and Zn-contaminated agricultural area in Pha Dei Village, Mae Sot District, Tak Province (N 16° 40΄ 35.9˝ E 98° 37΄ 37.4˝) at an altitude of 197 m for culture enrichment. To enrich Cd-resistant bacteria (BC), the first 5 g of each topsoil sample was added to 95 ml of nutrient broth (NB, 0.5% peptone, 0.3% meat extract, pH7.0) containing 50 or 100 ppm Cd chloride (CdCl2). After two weeks of consecutive incubation at 30oC, the bacteria were cultured on nutrient agar plates (NA, nutrient broth and 1.5% agar) supplemented with CdCl2 for 72 h at 30oC. The colonies of Cd-resistant bacteria were quantified as colony forming units per ml (CFU ml-1). The test biofertilizer (BF) was prepared under aerobic conditions, using enriched BC with rice bran supplemented with micronutrients and mineral additives to stimulate fermentation. The organic fertilizer (OF) was produced by fermenting the rice bran supplemented with micronutrients and mineral additives as mentioned in an aerobic environment, in absence of BC. The biofertilizers were stored at 4oC prior to use in greenhouse experiments. Hence, the treatments used in this study were listed in Table 1. The main components and bacterial compositions of each amendment are shown in Table 2.
For physicochemical analyses, properties of the biofertilizers as soil amendments or conditioners were determined: pH using a pH meter and OM content using wet oxidization and titration according to the modified Walkley-Black procedure (Nelson &Sommers 1996). Total contents of metal elements including Cd, Zn, Ca, Mg, S, Fe and Mn in biofertilizer samples were determined using microwave digestion and quantification using an atomic absorption spectrophotometer (Perkin Elmer AAnalyst 200).
Bacterial diversity and composition of the test biofertilizers compared with the enriched consortia were analyzed using 16S rRNA gene Illumina MiSeq sequencing as previously described (Seang-On et al. 2019). Total genomic DNA was extracted from 10 ml of the enriched culture and the biofertilizers were tested (three biological replicates per treatment) using QIAamp® DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturer instructions with some modifications. The 16S rDNA (V3-V4) bacterial primers containing the Illumina overhang adapter sequences (as underlined) 341F (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and 805R (5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) were used for PCR amplification (Herlemann et al. 2011). The PCR mixtures (25 µl) contained 12.5 µl of 2x KAPA HiFi Hot Start Readymix (KAPA Biosystems, USA), 5 µl of each primer (1 µmol l-1) and 2.5 µl of target DNA (5 ng µl-1). The PCR cycling conditions consisted of an initial denaturation step at 94oC (3 min), followed by 25 cycles of 98°C (20 sec), 55oC (30 sec) and 72°C (30 sec) and a final elongation at 72°C (5 min). The PCR products were cleaned-up on AMPure XP beads (Agencourt Bioscience, USA). The purified amplicons (550-bp fragments) were submitted to the Omics Sciences and Bioinformatics Center (Chulalongkorn University, Bangkok, Thailand) for paired-end sequencing on the Illumina MiSeq platform. Subsequently, the purified 16S RNA gene amplicons were then indexed using 2X KAPA hot-start ready mix and 5 µl of each Nextera XT index primer in a 50 µl PCR reaction, followed by 8 to 10 cycles of PCR amplification. The PCR cycling was set as aforementioned. Next, the indexed 16S RNA gene amplicons were purified on AMPure XP beads (Agencourt Bioscience, USA), pooled and diluted to a final loading concentration of 4 pM. Cluster generation and 250-bp paired-end read sequencing were performed on an Illumina MiSeq using the MiSeq Reagent Kit. Amplicon sequence analysis was performed with QIIME version 1.9.0.(Caporaso et al. 2010). All sequence reads were sorted based on their unique barcodes, trimmed for sequence quality and clustered at 97% identity for operational taxonomic units (OTUs). The UCHIME algorithm was used to discard chimera sequences (Edgar et al. 2011).
The microbial diversity index in terms of diversity (Shannon index) and richness (Chao1 index) were subsequently computed using MOTHUR (Schloss et al. 2009). To investigate the microbial composition and diversity, the Shannon diversity index, an estimator of species richness and diversity using a natural logarithm, accounts for both abundance and evenness of the taxa present, while the Chao1 richness estimator reflects diversity from abundance data and the number of rare taxa missed from under-sampling.
Greenhouse experimental design
All the experiments involving plants adhered to the relevant ethical guidelines on plant usage. Table 1 present the treatments used in this study. A 2,000 g soil sample was crushed, sieved (2-mm mesh size), and placed in a plastic pot as previously described with some modifications (Wang et al. 2019). Biofertilizers were mixed in long-term Cd contaminated soil at a rate of 3%. Hence, NPK basal fertilizer containing 0.25 g urea kg-1 soil, 0.15 g KH2PO4 kg-1 soil and 0.04 KCl kg-1 soil was initially dissolved in deionized water and thoroughly mixed with the soil in each pot. Subsequently, all pots were incubated with moisture at 75% of water holding capacity for five weeks to allow the nutrients in biofertilizers to be released in the soil, as well as promote the microbes in biofertilizers to work and to functionally act toward Cd stress. Thai rice seeds (PSL2) were sterilized with 5% hydrogen peroxide (H2O2) for 5 min, rinsed with distilled water and placed in a Petri Dish containing two pieces of filter paper. After germination, eight rice seedlings were transplanted in each plastic pot. The pots were arranged in a randomized complete block design with six replicates for each treatment. During rice growth, each pot was irrigated every three days with distilled water to maintain soil moisture at ca. 60 to 70% of water holding capacity. Greenhouse conditions were as follows: temperature 26 to 40oC, 55 to 70% relative humidity, 5,500 to 50,000 lx light intensity, and a 12/12 h photoperiod.
After 30 days of treatment, the roots and shoots were collected separately per biological replicate and stored at 4oC for measuring proline content, photosynthetic pigments and different enzymatic assays.
Four months after transplantation, the plant samples were washed with tap water, rinsed with deionized water several times until all excess soil was removed, and then the shoots and roots were harvested. Plant materials were oven-dried at 80oC for four days before determining weight. Soil material was collected from each pot and allowed to air-dry for five days. Soil and plant samples were subjected to chemical analyses.
Measurement of total protein content
The total protein content of rice leaves was quantified as previously described (Lowry &Rosebrough 1951). Plant leaves (0.5 g) were ground and added with phosphate buffer. The mixture was centrifuged at 3,000 rpm for 10 min. The resulting supernatant (0.1 mL) was added with distilled water to make the volume up to 1 mL. This solution was added with the equal volume of alkaline CuSO4 reagent and shaken for 10 min. Finally, the Follin reagent was added and then incubated for 30 min at 28 ± 2 oC. Readings were measured at 650 nm. Bovine serum albumin (BSA) was taken as a reference for the calculation of total protein contents.
Estimation of photosynthetic pigments
Photosynthetic pigments (chlorophyll (Chl) a, b, and carotenoids) of rice leaves were estimated as previously mentioned (Burnison 1980). Plant leaves (0.5 g) were added with 10 ml of dimethyl sulfoxide (DMSO) and then heated at 65oC in water bath for 4 hrs. The supernatant was separated, and its absorbance was recorded at 663 nm, 645 nm, for Chl a, Chl b, and 480 nm for carotenoids, respectively.
Estimation of proline content
Proline contents were determined by using previous protocol (Bates et al. 1973). Rice leaves (0.5 g) were ground in 80% ethanol and then heated at 80oC for 1 hr in a water bath. After centrifugation, 0.5 ml supernatant was taken into a new test tube, added with 0.5 ml dH2O and 1 ml of 5% phenol, and placed in an incubator for 1 hr. After incubation, 2.5 ml sulfuric acid was added and the readings were measured at 490 nm.
Determination of enzymatic antioxidant activities
Enzyme extracts
For preparing enzyme extracts, 0.5 g leaves and roots were ground in 3 ml phosphate buffer (pH 7.8) and subjected to homogenization on ice. The solution was made to 5 ml and centrifuged at 13,000 rpm for 20 min at 4oC. The supernatant was covered with aluminum foil to avoid light exposure and stored at 4oC for subsequent enzyme assays.
Ascorbate peroxidase (APX) activity
APX (EC# 1.11.1.11) activity was quantified by examining the rate of ascorbate oxidation at a wavelength of 290 nm as the previous method (Asada 1987). The reaction mixture consisted of 50 mM phosphate buffer pH 7.0, 0.1 mM H2O2, 0.5 mM ascorbic acid, and 100 µl of enzyme crude extract.
Superoxide dismutase (SOD) activity
SOD (EC# 1.15.1.1) activity was subjected to assess the inhibition in the photoreduction of nitro blue tetrazolium (NBT) as previous procedure (Beyer Jr &Fridovich 1987) . Reaction mixture was taken with 50 mM sodium phosphate buffer (pH 7.6), 0.1 mM EDTA, 50 mM sodium carbonate, 12 mM L-methionine, 50 µM NBT, 10 µM riboflavin, and 100 µl of enzyme crude extract. For comparison, a set of reactions with all components except the crude extract was taken as control. To start the reactions, the reaction tubes were exposed to white light for 15 min. Reactions were terminated by switching off the lights and readings were recorded at 560 nm.
Sampling and metal analyses of soil and plant tissue
Total and extractable contents of metal elements in soil and plant samples were determined using microwave digestion and diethylenetriamine pentaacetate (DTPA) extraction, respectively. Total (strong acid-extractable) Cd and Zn in soil before and after planting was estimated by digesting approximately 1 g of air-dried soil with 4.5 ml of 37% hydrochloric acid (HCl), 1.5 ml of 65% nitric acid, and 1 ml of 30% H2O2 in a microwave digestion system (Milestone ETHOS One, USA). A similar procedure was employed to digest plant materials, but without HCl addition. The amount of DTPA-extractable Cd and Zn in soil was determined using 0.005M DTPA+0.01 M CaCl2+0.1M triethanolamine, pH 7.30 at a soil-to-solution ratio (w/v) of 1:2. The metal concentrations in both the digests and extracts were quantified using an atomic absorption spectrophotometer (Perkin Elmer AAnalyst 200).
Statistical analyses
Data were subjected to statistical analysis using two-way ANOVA (SPSS Software) to detect significant differences with 95% confidence level (P-value ≤0.05).