The study was conducted at the nursery of the School of Industrial Technology, USM, Pulau Pinang from November 2010 to February 2011. The average temperature ranged between 30°C and 31°C in the nursery. The average annual rainfall was 2,670 mm, and relative humidity was 70% to 90%.
Decanter cake obtained from Malpom Industries Sdn Bhd, Nibong Tebal, Pulau Pinang, Malaysia was dried, powdered, and passed through a 2-mm sieve. Similarly, soil that was dug up from about 10 cm deep and was taken from a nearby land was dried, homogenized, and passed through a 2-mm sieve.
The experiment consisted of planter boxes in a completely randomized design of about 25 cm in diameter and 15 cm in depth. Treatments in the ratios of 10%, 20%, and 30% (w/w) DC with unamended soil (0%) as control were thoroughly mixed and filled into the planter pots. Identical water regime was maintained and then left for 15 to 20 days for stabilization and mineralization of the organic matter. After the stabilization period was over, soil was collected in triplicates for physicochemical analysis. Prior to sowing, seeds of A. esculentus were soaked in distilled water for 3 min. About seven to eight seeds per treatment were sown manually at equidistant positions. After germination, the plants were thinned to three per pot, and identical temperature and humidity conditions were provided throughout the growth period.
Measurement of data
Soil samples of each treatment were collected in triplicates, crushed, air-dried, and sieved with a 2-mm mesh size. Decanter cake samples were also taken for further physicochemical analysis. The pH of samples was measured using a pH meter (Hach, sensION3, Loveland, CO, USA) in a 1:10 (w/v) suspension, while the electrical conductivity (EC) in 1: 2 (w/v) was measured using the Hach sensION5 conductivity meter. A CHN analyzer (2010 PerkinElmer Instruments, Branford, CT, USA) was used to determine the C/N ratio of the amended soils. Bulk density test was carried out by taking 10 g of soil sample that was oven-dried at 105°C, slowly poured into a measuring cylinder, and gently tapped for compaction (expressed in g cm-3) (Radojevic and Bashkin 2006).
Specific gravity test was carried out, according to Radojevic and Bashkin (2006), by taking 10 g of dry sample in an empty glass bottle; the weight was taken thereafter. The bottle was then rinsed and filled to the brim with water, and the weight was taken. The formula used to calculate the specific gravity for the soil samples is as follows:
where M 1 = weight of empty glass bottle (g), M 2 = weight of bottle and soil (g), and M 3 = weight of bottle and water (g).
For analysis of metals in the soil and decanter cake samples, wet digestion method was followed: 1 g of air-dried soil sample was digested in 20 ml of triacid mixture (HN03:H2S04:HCLO4) for 8 h at 80°C till transparency. The digested solution was then made up to 50 ml with deionized water and filtered with a Whatman no. 1 filter paper. The filtrate was analyzed for various metals using an atomic absorption spectrophotometer (PerkinElmer Analyst 200 AAS 2007).
Morphological and growth parameters
Plant samples with intact roots were randomly taken from each treatment 65 days after sowing (DAS) and washed with running distilled water to remove soil particles that adhered on the roots. Growth parameters such as shoot length, root length, leaf area, and number of leaves were measured (Singh and Agrawal 2007, 2009). The leaf area was measured by a portable laser leaf area meter (Leaf area meter CI 203, CID Bio-Science, Camas, WA, USA). For biomass determination, separate root, shoot, and leaf parts were placed in the oven at 80°C until a constant weight was reached. The plant parts were then weighed separately, and biomass accumulation was expressed as grams per plant. Total plant biomass was estimated as the total dry weight of plant parts. Yield is expressed as fresh weight of fruits per plant at the time of harvest. For growth indices, such as leaf area ratio (LAR), leaf weight ratio (LWR), specific leaf area (SLA), and root shoot ratio (RSR) formulae by Hunt (1982), were used as shown:
Photosynthesis pigments (chlorophyll and carotenoid) in fully expanded fresh leaves sampled at 45 DAS was estimated using the Machlachlan and Zalik (1963) and Duxbury and Yentsch (1956) methods. Phenol content was determined in accordance with the method of Bray and Thorpe (1954), and thiol content was estimated using the method of Fahey et al. (1978). Ascorbic acid concentration in fresh leaves was determined using the method of Keller and Schwager (1977).
Data obtained from the experiments were subjected to a one-way ANOVA test using the SPSS 18 software (SPSS Inc., Chicago, IL, USA; SPSS, Richmond, VA, USA) to assess the significance of differences and biochemical, physiological, and growth parameters of DC in comparison with those of the control (unamended soil). Mean values were separated using Tukey's test at 5% probability level.