Soil Amendments with Sugarcane Bagasse and its Effect on Soil Humic Acid Contents and Chinese Cabbage Growth Components

The population increase and industrial development produce an enormous amount of organic residues creating great environmental problems now a day. The appropriate agriculture use of these residues can become advantageous for the mankind because it allows nutrients recycling, improve crop production, less pollution problems and as well the improvement of the physical, chemical and biotic conditions of the soil [1]. Organic matter applied to the soil favors the development and growth of plants because it prevents nutrients loss by leaching [2-4]. The sugarcane is a grassy crop that produces, in a short period, a high income of biomass, energy and fibres, being considered one of the plants with larger photosynthetic efficiency [1]. Its plantation in a wide scale is traditional in several countries of the tropical and subtropical regions to produce sugar, alcohol and other bioproducts. Several tons of sugarcane residues are produced and need to be conditioned. Experiments have been conducted to study the viability of those residues on fish feeds, as nematicide on mandarin culture as horticulture substrate mixes or wheat production [5-8]. Sugarcane bagasse is a source of vegetal fiber that has potential use on the industry of polymeric composites [9]. Sugarcane bagasse (SCB) produce humic acid (HA) after decomposition which have colloidal character and huge active surface which provide fine adsorptive properties [10]. Adsorptive features of HA allow to supply necessary micro-and macronutrients to plants and eliminating from the ground ionic and molecular impurities in the form of heavy metals [10,11]. All these potential uses have the major goal of resolving the disposal problems of sugarcane residues. The main objective of this study is to quantify the amount of HA produced after amending the soil with SCB with respect to the entire life span of growing plant Chinese cabbage (Brassica rapa, subsp. pekinensis). Abstract


Introduction
The population increase and industrial development produce an enormous amount of organic residues creating great environmental problems now a day. The appropriate agriculture use of these residues can become advantageous for the mankind because it allows nutrients recycling, improve crop production, less pollution problems and as well the improvement of the physical, chemical and biotic conditions of the soil [1]. Organic matter applied to the soil favors the development and growth of plants because it prevents nutrients loss by leaching [2][3][4]. The sugarcane is a grassy crop that produces, in a short period, a high income of biomass, energy and fibres, being considered one of the plants with larger photosynthetic efficiency [1]. Its plantation in a wide scale is traditional in several countries of the tropical and subtropical regions to produce sugar, alcohol and other bioproducts. Several tons of sugarcane residues are produced and need to be conditioned.
Experiments have been conducted to study the viability of those residues on fish feeds, as nematicide on mandarin culture as horticulture substrate mixes or wheat production [5][6][7][8]. Sugarcane bagasse is a source of vegetal fiber that has potential use on the industry of polymeric composites [9]. Sugarcane bagasse (SCB) produce humic acid (HA) after decomposition which have colloidal character and huge active surface which provide fine adsorptive properties [10]. Adsorptive features of HA allow to supply necessary micro-and macronutrients to plants and eliminating from the ground ionic and molecular impurities in the form of heavy metals [10,11]. All these potential uses have the major goal of resolving the disposal problems of sugarcane residues. The main objective of this study is to quantify the amount of HA produced after amending the soil with SCB with respect to the entire life span of growing plant Chinese cabbage (Brassica rapa, subsp. pekinensis).

Mode of collection and preparation of soil and sugarcane bagasse samples
The composite sampling method was used for sampling, where about 25 subsamples of each 2kgs collected from Ngongona villege in Dodoma region and a composite sample of 50kgs used in this study. Samples were collected with the help of an auger from the 10cm depth and kept in plastic bags and packed in the bucket [12]. Soil samples were air dried on dry wood which act as drying surface. 20kgs of sugarcane bagasse was collected from local sugarcane juice vendors (Ngongona sugarcane juice extracts) manually by using hands processed before use. The sugarcane sample was air dried for four days and then dried in an oven at 70°C for two consecutive days. Finally grounded by using blender and sieved (2mm mesh) to make fine powder [13].

Extraction of humic acid
SCB amended (50:50) soil (10g) was mixed with 5mL of distilled water allowed decomposing at 37 °C. Four samples were prepared in the same manner and allowed to decompose at different periods (0, 10, 20 and 30 days). Humic acids were extracted using the classic alkali/acid fractionation method [14]. The SCB amended soil was digested in 0.1 N KOH (1:10 W/v) for 24 hours at room temperature (23 ± 2 °C). The undigested bulk residue from SCB compost was then separated from the solute fraction by centrifugation at 5000rpm for 30 minutes followed by vacuum filtration through a glass filter paper. The filtered supernatant was acidified to pH 2 with 6.0 N H 2 SO 4 and kept in a cold room in the dark for 24hrs in order to obtain flocculation of humic acids. After acidification, the humic precipitate (humate) was collected by centrifuging at 5000rpm for 30min, washed three times with distilled water to remove residual H 2 SO 4 , freeze-dried and brown powder was obtained for each treatment which was used for FT-IR to characterize humic acid. The experiment was carried out at College of Natural and Mathematical Sciences, The University of Dododma, Dodoma, Tanzania from Mid-May to July 2018. This experiment employed quasi-experimental method using four treatments. Each treatment has potted plants composed of three sample plants per pot (3.5kg) in triplicate making a total of 12 pots. The collected soil was mixed thoroughly to form homogeneous mixture in different concentrations (0, 2, 5 and 10%) of SCB (Table 1). For proper decomposition, SCB was applied one month prior to seed planting in the field since the rate of decomposition is affected by temperature, moisture, population and diversity of soil microorganisms [15].

Field management
Chinese cabbage seeds were germinated following the recommended practice in the soil with different treatments (Table  1). One pot has three sample plants. Plants were watered with 100mL for the first 3 days to check the response of pot treatments. Additional 50mL of water was administered, depending on the response of the plants to respective pot treatments ( Table 2), but equal amount of water (150mL) was administered for every plant, using graduated cylinder. To control pests and diseases, the use of contact insecticide (cypermethrin) available in the market was done. It was sprayed following the prescribed dosage (2mL per a liter of water) of the manufacturer.

Data collection and analysis
Agronomy and physiological data of Chinese cabbage growth were collected as indicated below for different treatments ( Figure  1) and analyzed by Microsoft excel.

Agronomic parameters
Days to emergence: This parameter of the Chinese cabbage was determined by counting the number of days from sowing to the time when 50% of the plants started to emerge the tip of panicles through visual observation.
Plant height and root length: Plant height was measured at maturity from the ground level to the top of the Chinese cabbage from each treatment and for selected three plants and the average was taken as plant height (cm). Root length also studied to the same plant for which plant height was determined.

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Number of leaves per plant: Number of leaves were counted from three representative plants from each pot and averaged as per plant.

Physiological parameters
Plant samples were collected at 5 weeks after sowing. Chinese cabbage plants were separated into leaves and roots for determining relative water content, plant dry mass, root to shoot ratio.

Relative water content (RWC):
Fully expanded youngest leaves were selected from different plants from each treatment ( Figure 2). Five leaves were sampled and weighed immediately to determine the fresh weight (FW). Then immersed in distilled water in Petri-dishes for 24h in darkness and then turgid weight (TW) was determined. The leaves were dried in an oven at 70 °C for 24h and the dry weights (DW) were obtained [16]. From the obtained data, relative water content was calculated using the following formula: Root to shoot ratio: The above ground part (shoot) and below ground part (root) of Chinese cabbage from each treatment were sampled from the plant which is grown very well. The roots were washed in tap water to remove the soil particles and blotted to dry on paper towels. The fresh weight was determined immediately after harvesting using an electronic weighing balance. The collected samples of shoots and roots were dried in an oven at 70 °C for 24 hours to obtain dry weight. Then root to shoot ratio was calculated by using formula below [17].

R
Weight of dry root Root to shoot ratio ratio S Weight of dry shoot

Results and Discussion
The amounts of humic acid extracted from SCB after decomposition at different periods were shown in Table 3. Higher amount (0.2779g) of HA was extracted after 30 days, and small amount (0.0053g) was extracted from 0 day of SCB decomposition. The amount of HA extracted from soil with SCB is significant with respect to the time taken for decomposition of SCB. The humic acid content of soil with SCB is proportional to the degree of decomposition of SCB [3]. Characterization of humic acid using FTIR spectra Figure 3 shows the FTIR spectra of the humic acid samples collected at 0, 10, 15, 30 days. In general, all spectra are almost similar in the position of the main bands, but some differences can be observed in their relative intensity. All the spectra showed bands that could be assigned to the main groups, as: 3371.74 -3335.45cm -1 the intense and broad absorption band due to O-H bond stretching which mainly belongs to the carboxylic acids involved in hydrogen bonding. The week and narrow bands at 2930.99-2854.66cm -1 for HA extracted from zero and ten days after decomposition of SCB was due to symmetric stretching bands of aliphatic C-H bonds of -CH 3 , -CH 2 -and tertiary C-H. The obtained absorption band is very similar to that from soil HA which is reported by Stevenson [3]. It is possible to differentiate the source and the humification condition of the organic matter by using FT-IR spectrum. The HA with relatively low degree of humification has a spectrum with -CH 3 and -CH 2 -and tertiary C-H absorption bands (generally located around 2900-2850cm -1 ). It is clearly observed that these bands are absent or overlapped with O-H band of COOH group in the spectra of HA samples collected at 15 and 30 days of decomposition of SCB. Moreover, the band intensity of carboxylic O-H groups increased with the number of days of decomposition of SCB. This implies as the decomposition period increases the number of carboxylic acid groups increased. Hence it is proved that more humic acid produced with more number of days of decomposition of SCB. The sharp and medium intense bands (1633.79, 1706.76, 1635.62 and 1636.23cm -1 for 0, 10, 15 and 30 days respectively) observed in the carbonyl region also indicates the production of humic acid with the decomposition of SCB. A pair of bands observed between 1598.11 -1450cm -1 (for C=C) indicate the presence of aromatic ring in humic acid. The results were slightly similar with the results reported by Reddy and his co-workers (2018) on FTIR spectrum of HA of paddy soils [18].

Effects of SCB on agronomic parameters of Chinese cabbage
Days to seed emergency: Days to emergence generally took 3 to 5 days. The plants emerged fast from the soil mixed with 10% of SCB (Treatment B 3 ). Faster germination from B 3 treated pot might be due to SCB amendment germination. In order to emerge the plants from 50% of seeds it took only three days in the case of soil with 10% of SCB whereas it took five days in the case of soil without any SCB (control). With the increase in the percentage of SCB from 0 to 10% the plant emergence from 50% of seeds took a smaller number of days ( Table 4). The absorption of humic substances produced by SCB into seeds has a positive influence on seed germination and seedling development. Availability of humic (HA) or fulvic acids (FA) to seeds will increase the seed germination, resulting in higher seed germination rates [19]. Results of this study are similar with the findings of Eyheraguibel [20]. According to their study the germination was started 3 days after sowing and the first daily count of germinated seed showed more radicle emergence from the soil with humic like substances. Stehouwer and Macneal [21] recorded an increase of germination and fescue seedling establishment after the first leaching event as a response of the salt decrease following compost amendment [21]. Plant height and root length: Plant height increased from 5.3 to 15.3cm with the increase in the percentage of SCB in the soil from 0 to 10%. This indicates the increase in growth of the plant in soil amended with SCB. This might be due to increase in the amount of humic acid and organic carbon in the soil after amendment with SCB. The tallest root length (6.2cm) was observed in the soil amended with 10% of SCB (treatment B 3 ) and the shortest root length (2.2cm) was obtained from the control (Table 4). For treatment B 3 the amended organic matter improves the physical properties of the soil and causes increase of root development which helps the uptake of more water and nutrients [22]. Both plant height and root length increased significantly with the increase in the percentage of SCB in the soil. The significant increase might be from soil properties that support the root growth due to the enough oxygen diffusion to the root tip and supply of enough water for root growth. The results of this study are like that of reported by Robert & Ronny [22] that after application of organic matter, soil sodium adsorption ratio declines 56%, and root length of plant increases 140%. Sodium induces soil structural deterioration (slaking, aggregate destruction, and clay and organic colloid dispersion), leading to subsequent water infiltration and percolation problems [22].

Number of leaves in plant:
The average number of leaves per plant was found highest (8.7) in the soil with 10% of SCB and lowest (3.6) was in the control ( Table 4). The average number of leaves of Brassica rapa, subsp. Pekinensis growing in soil with different concentration of SCB reveals an overall increasing pattern from control to B 3 (10% SCB). This might be due to improvement in the soil properties to increase soil fertility, water holding capacity and soil porosity etc. to support the plant growth. SCB when applied to land increases soil fertility by providing macro nutrients such as nitrogen and phosphorous and micronutrients such as Zn and Cu [22]. Relative water content: Relative water content (RWC) is the most appropriate measure of plant water status in terms of the physiological consequence of cellular water deficit. Water potential as an estimate of the energy status of plant water is useful in dealing with water transport in the soil-plant-atmosphere continuum. However, it does not account for osmotic adjustment. For the same leaf water potential two different cultivars can have different leaf RWC, indicating a corresponding difference in leaf hydration, leaf water deficit and physiological water status. Hence RWC is an appropriate estimate of plant water status in terms of cellular hydration under the possible effect of both leaf water potential and osmotic adjustment [23]. From the data in Table 5, relative water content increased significantly from 57.29 to 73.21, when SCB increased from 0 to 10% in the soil (treatments C (0% SCB) to B 3 (10%SCB)).

Plant dry mass and fresh weight:
Results presented in Table  5 shows that the fresh weights of root and shoot (g per plant) significantly increased with increase in the SCB content in the soil from 0 to 10% (treatments from C, B 1 , B 2 and B 3 respectively). Furthermore, dry shoot and root biomass of the plant were significantly enhanced in the soil with SCB as compared to the soil without SCB (control). The root/shoot ratio also enhanced due to soil amendment with SCB comparing with the control (Figure 4). The observed enhancement of plant growth by the application of SCB to the soil is due to increase in uptake of the elements such as N, P, K, Fe, Zn, and Mn nutrients [24]. Moreover, enhancement of photosynthesis, plant root respiration has resulted in greater plant growth with HA produced by SCB [25]. The performance of the soil in the plant growth after the application of SCB was due to enhancement in moisture retention and the improvement of nutrients supply in the root zone [26].

Conclusion
Sugarcane bagasse is generally considered as an agricultural waste product; however, the present findings show it contains enough amounts humic acid after decomposition. Application of fertilizer can save chemical fertilizers along with minimizing environmental pollution. By comparing the levels of sugarcane bagasse application in this study, 10% was suggested to be the standard dose due to best yield parameters such as the root and shoot length of plant, root and shoot dry weight of plant, number of leaves and relative water content of Chinese cabbage (Brassica rapa pekinensis) crop in the soil amended with SCB.