Wednesday, March 26, 2014

Influence of biochar in seed germination and growth rate (part 1)

Biochars affects seed germination and fast growth of seedlings
     Biochar has been reported to both increase (Chan et al. 2008; Yamato et al. 2006) and decrease (Deenik et al. 2010) plant growth and yield but there have been few studies reporting the influence of biochar on early stages of plant growth such as on seed germination and seedling growth. The stimulation or inhibition of seed germination due to biochar application has mostly been investigated for forest plants (Choi et al. 2009; Pierce and Moll 1994; Reyes and Casal 2006; Tian et al. 2007).

    For agricultural plants,activated charcoal (steam treated) enhanced seed germination of potato (Bamberg et al. 1986) while Van Zwieten et al. (2010) showed that wheat seed germination was increased with a single dose (10 t/ha) of paper mill biochar. In contrast, Free et al. (2010) reported that maize seed germination and early growth were not significantly affected by biochars made from a range of organic sources.

       The application of biochar to soil can alter organic matter mineralization (Steiner et al. 2008; Wardle et al. 1998) which is linked to the release of nutrients such as nitrogen (Manzoni et al. 2008; Murphy et al. 2003). The resultant change in nutrient status of the soil may affect both seed germination and seedling growth.

     Application of biochar to acidic soils can increase soil pH to alkaline levels, especially if higher rates of biochar are applied and changes occur to soil cation exchange capacity (CEC) (Ogawa 1994). The diversity in characteristics of biochar indicate that biochar responses will depend on the type and rate of biochar applied to soil as well as on soil characteristics such as soil C, pH, CEC and other components of soil fertility.

pH and EC
The pH and EC of biochar were measured in water at 1: 5 (w/v) ratios. Soil pH was also measured in CaCl2 at 1:5 (w/v) ratios. 

Soil-less Petri dish bioassay 
     15 (zea maiz) seeds were sown in Petri dishes (8.5 cm diameter) on a layer of filter paper moistened with deionized water.  20 mL of DI water was added to the Petri dish for each rate of biochar. Each of the four biochar types was added at the rates 0, 0.5, 1.0, 2.5, 5.0 g/Petri dish (equivalent to 0, 10, 20, 50, 100 t/ha on a volume basis at 10 cm soil depth) with three replicates following the design recommended by Morrison and Morris (2000) where an individual Petri dish was considered as a replicate and a control treatment was used for each biochar.

Rates of biochar to be applied in germinations
     All Petri dishes were covered with lids and incubated in the dark at 25°C for 72 h after germination percentage and root length was assessed. Root length of germinated seeds was measured in fresh roots using a ruler, and summed for each Petri dish (m/Petri dish). 

Preparation of germination in petri dish

     Effect of biochar at different rates 0, 0.5, 1.0, 2.5, 5.0 g/Petri dish (equivalent to 0, 10, 20, 50, 100 t/ha based on10 cm field depth) on seed germination average conducted in the soil less Petri dish bioassay.

Petri dish bio-assay

Coconut fiber

Palm fiber

Criptomeria japonica

    Most of the biochars used in these experiments were alkaline (pH in water 8.1 to 9.11). TDS: for ppt, ppm and EC, coconut fiber biochar showed the highest contents. Being eucalyptus biochar the one with the lowest levels.

Growth rate
     Biochar type and application rate influenced wheat seed germination and seedling growth in the soil-less Petri dish and soil-based bioassay. Germination in coconut fiber, eucalyptus and palm fiber showed little difference among them, being criptomeia japonica the biochar with less seeds germinated.

     Fig 1). Early root growth of corn seeds was different in response in eucalyptus biochar compared to the results of the other three biochars (figures 2,3,4 and 5).

Germination average (Figure 1)

Palm fiber (Figure 2)

Coconut fiber (Figure 3)

Eucaliptus   (Figure 4)

Criptomeria japonica (Figure 5)

ICP of biochars (Figure 6)

     In conclusion, the four biochar types used in this study generally increased wheat seed germination at rates of application <10 and <50 t/ha the rest of the treatments tended to inhibit germination at the highest rate of application under the bioassay conditions.

    This investigation supports the proposal of Major (2009) that a germination test could be a useful screening process for evaluating biochars. However, it is important to use several rates of biochar in the bioassay because of differences in response observed in this study. ICP analyses (fig.6) determined the contents of various elements in the biochars showing similarities in Fe, Pb, Zn, Cr, Na, and Cu, with differences in K, Ca, and Mg.

    We recommend the soil-less Petri dish bioassay as a preliminary ecotoxicological test for biochar screening because it is rapid and simple, and it avoids the need for use of a ‘standard’ soil which is difficult to collect, transport and maintain across quarantine boundaries. Finally, it is recommended that toxicity bioassays for biochar are repeated (in addition to the replication used within each test) to ensure reproducibility.

Table with all the information showed in graphs


Bamberg JB, Hanneman RE Jr, Towill LE (1986) Use of activated charcoal to enhance the germination of botanical seeds of potato. Am Potato J 63:181–189

Chan KY, Van Zwieten L, Meszaros I, Downie A, Joseph S (2008) Using poultry litter biochars as soil amendments. Aust J Soil Res 46:437–444

Choi D, Makoto K, Quoreshi AM, Qu LY (2009) Seed germination and seedling physiology of Larix kaempferi and Pinus densiflora in seedbeds with charcoal and elevated CO2. Landsc Ecol Eng 5:107–113

Deenik JL, McClellan T, Uehara G, Antal MJ, Campbell S (2010) Charcoal volatile matter content influences plant growth and soil nitrogen transformations. Soil Sci Soc Am J 74:1259–1270

Free HF, McGill CR, Rowarth JS, Hedley MJ (2010) The effect of biochars on maize (Zea mays) germination. New Zeal J Agr Res 53:1–4

Major J (2009)A guide to conducting biochar trials—International Biochar Initiative.pp1-30, (

Manzoni S, Jackson RB, Trofymow JA, Porporato A (2008) The global stoichiometry of litter nitrogen mineralisation. Science 321:684–686

Morrison DA, Morris EC (2000) Pseudoreplication in experimental designs for the manipulation of seed germination treatments. Austral Ecol 25:292–296

Murphy DV, Recous S, Stockdale EA, Fillery IRP, Jensen LS, Hatch DJ, Goulding KWT (2003) Gross nitrogen fluxes in soil: theory, measurement and application of 15N pool dilution techniques. Adv Agron 79:69–119

Ogawa M (1994) Symbiosis of people and nature in the tropics. Farming Japan 28:10–34

Pierce SM, Moll EJ (1994) Germination ecology of 6 shrubs in fire-prone cape fynbos. Vegetation 110:25–41

Reyes O, Casal M (2006) Seed germination of Quercus robur, Q-pyrenacia and Q-ilex and the effects of smoke, heat, ash and charcoal. Ann Forest Sci 63:205–212

Steiner C, Das KC, Garcia M, Förster B, Zech W (2008) Charcoal and smoke extract stimulate the soil microbial community in a highly weathered xanthic Ferralsol. Pedobiologia 51:359–366
Tian YH, Feng YL, Liu C (2007) Addition of activated charcoal to soil after clearing Ageratina adenophora stimulates growth of forbs and grasses in China. Tropical Grasslands 41:285–291

Van Zwieten L, Kimber S, Morris S, Chan KY, Downie A, Rust J, Joseph S, Cowie A (2010) Effects of biochar from slow pyrolysis of papermill waste on agronomic performance and soil fertility. Plant Soil 27:235–246

Yamato M, Okimori Y, Wibowo IF, Ashori S, Ogawa M (2006) Effects of the application of charred bark of Acacia mangium on the yield of maize, cowpea and peanut, and soil chemical properties in South Sumatra, Indonesia. Soil Sci Plant Nutr 52:489–495

Sunday, March 23, 2014

Advantages of using municipal solid waste incineration bottom ash in combination with biochar mixtures as soil modifiers.

Bottom ash and biochar
  For this experiment, Different BA was obtained from a BA recycling facility located in Southern Taiwan.

YING CHENG COMPANY -  where incineration bottom ash is treated

     Bottom ash from three different cities (Pingtung, Chiayi and Chunghua City) was collected and was air dried for 3 days at room temperature. Then it was sieved using two mesh sizes (mesh 1- 19.10 mm and mesh 2- 4.700 mm). For the effect of sieve size two different mesh sizes were chosen, according to Skorupskaitė and Denafas (2004) the all kinds of ash particles, especially in the area of small particles, have a relatively big surface area, porous surface, and for this reason they could have a huge absorptions capacity.

  Two different feedstocks were used to produce the biochars used in this report: rice husks and bamboo, each material was generated at different temperatures. The rice husks from the International Rice Research Institute (IRRI) from the Philippines were generated at 400ºC, and the rice husk biochar from the Asahi Company (Taiwan) was generated at 500ºC. The Bamboo Biochar from the Industrial Technology Research Institute (ITRI) was generated at both 300ºC and 600ºC. All biochars were obtained by pyrolysis.


The used biochar samples were named as “rice husk 400”, “rice husk 500”, “bamboo 300” and “bamboo 600”. All biochars were made by pyrolysis. Each biochar was mixed separately with soil. Each one of the bottom ashes was based on an equivalent rate of 10 t ha−1, assuming incorporation of the biochar to 50 mm soil depth.  Soil without biochar and pure bottom ash was also tested in this experiment. Mixing was achieved manually.

We aim to determine which of the biochars, in combination with bottom ash with and without the application of fertilizer, provides the best results in terms of plant growth, root yield and dry biomass weight of Zea mays L.

Plant material
    For this experiment, we used glutinous corn (Zea mays L.) seeds. In Taiwan, corn is mainly grown for both human consumption and as feed for livestock. It can be harvested after 70-90 days.  Germination takes place at around 7 days. The temperature required for germination is between 20°C-25°C. Full sunlight and mild weather are also required.

Germination, growth test, root elongation and treatments pH
  The bottom ash from the different facilities was tested with and without the addition of fertilizer. Initially a germination test was conducted; thirty maize seeds (Zea mays L.) were sown into germination trays using one tray for each of the different test treatments (with fertilizer and without fertilizer). 

    Each biochar was mixed separately with soil and bottom ash. Trays were filled with either soil or soil-biochar-bottom ash mixtures, randomly placed on net house benches and watered before sowing the seeds. Trays were watered daily. Germination percentages were recorded between days 5 and 10 after sowing. Data is presented only for the 7th day of sowing corresponding to peak germination.  For each bottom ash test, 7 pots were used with 4 replicates each (n=4) using the following mixtures: 1- soil, 2- bottom ash + soil, 3- bottom ash + rice husk 400, 4- bottom ash + rice husk 500, 5- bottom ash + bamboo 300, 6- bottom ash + bamboo 600 and 7- bottom ash. Consequently plant growth test was performed. Pots were prepared and seeds were sown at a depth of 2cm. Water was applied after the sowing. Fertilizer (N-P-K) was added after 2 days of germination.

     Plant morphology was observed, and every week plant size was measured. Plants were harvested after one month. They were cleaned, and washed with DI water. Excessive water was removed to later obtain the total weight. Plants were later separated from roots, these ones were measured to obtain root elongation, later were air dried and separated into stem, roots and leaves.

Analysis of metal elements
    Heavy metal analysis (ICP) was carried out to identify the properties of the different bottom ashes used. The leaching extraction procedure followed USA EPA method # 1311 with minor modifications (EPA, 1990). Five grams of ground and weighed bottom ash were put in a volumetric flask together with 1000 ml of distilled water and 5.7 ml of acetic acid. Samples were left for 18 h in a toxicity characteristics leaching procedure (TCLP) rotator. After this procedure, samples where filtered and analyzed through a Perkin-Elmer 3000-XL inductively coupled plasma (ICP-AES) spectrometer.

Determination of total phenol content
    After harvesting, the plants were washed, cut into small pieces and dried in an oven at 65°C for 72 h. The dried material was then ground and passed through a 250-µm sieve mesh. The powder obtained was packed in polyethylene bags and stored in a refrigerator at 4°C for future use. The total phenolics were determined according to the Folin-Ciocalteau method (Rossi, 1965; Waterhouse, 2002; Koffi et al., 2007). Approximately 2 g of ground plant powder was mixed with 8 ml of solvent (acetone), and 2 mL of water was added. The tube was capped and shaken at 200 rpm for 30 min in a water bath at 60oC (gyratory water bath shaker, Model G76D, New Brunswick Scientific Co., Edison, NJ). The tubes were removed, vortexed and centrifuged at 2,000 rpm using a Dynac II centrifuge (Becton & Dickinson Company, Franklin Lakes, NJ) for 2 min.

Folin-Ciocalteau method


    The samples were filtered through a 0.45 mm Millipore syringe filter (Whatman, Inc., Clifton, NJ). The total phenolics in the filtrate were determined colorimetrically. A 0.2 N Folin-Ciocalteau reagent (Sigma) was freshly prepared by diluting a 2 N stock solution with water. A volume of 100 mL of filtrate was added to 900 mL of distilled water, and 5 mL of 0.2 N Folin-Ciocalteau reagent was mixed. Saturated sodium carbonate (Sigma Chemical Co.), 4 ml of a 75 g/L solution, was added, followed by mixture with a vortex. The tubes were incubated for 2 h at 25°C. Then, the absorbance was read at 750 nm with a UV/VIs-105 Genesys spectrophotometer (Thermo, USA). The total phenolic content of the samples were calibrated using catechins mono-compound and was expressed as parts per million and converted to (mg/L). All measurements were performed in duplicate.

2nd part of the experiment:
Municipal solid waste incineration bottom ash and biochar from binary mixtures of organic waste as agronomic materials

Bottom ash from municipal solid waste
     The composition of municipal solid waste varies over time and from country to country, due to the differences in lifestyle and waste recycling processes of a country; the ash content will vary. Generally, the chemical and physical characterization of ash depends on the compositions of the raw MSW, the operational conditions and the type of incinerator and air pollution control system design (He et al., 2004). 

    The chemical composition shows that the major elements are Si, Al, Fe, Mg, Ca, K, Na and Cl. Furthermore, SiO2, Al2O3, CaO, Fe2O3, Na2O and K2O are common oxides found in ash SiO2, the most abundant compound that exists in MSWI bottom ash, up to 49% in content. 

    For heavy metals, Cr, Cu, Hg, Ni, Cd, Zn and Pb are the most common to be found in MSWI ash, with Zn and Pb usually constituting the largest amounts. These metals may cause leaching problems and are harmful to the environment without proper treatment (Lam et al., 2010).

    Prior to planting in pots, a germination test was performed.  Thirty maize seeds (Zea mays L.) were sown into germination trays using one tray for each of the different test treatments (M1+F=Mesh 1 with fertilizer, M1/wF=Mesh 1 without fertilizer, M2+F=Mesh 2 with fertilizer and M2/wF= Mesh 2 without fertilizer). Each biochar was mixed separately with soil and bottom ash. Trays were filled with either soil or soil-biochar-bottom ash mixtures, randomly placed on net house benches and watered before sowing the seeds. Trays were watered daily. Germination percentages were recorded between days 5 and 10 after sowing.

    Data are presented only for the 7th day of sowing corresponding to peak germination. For the plant growth test, one pot was used for each of the binary mixtures. In total, seven pots were used for each one of the four treatments for the three different locations (M1WF=Mesh 1 with fertilizer, M1NF=Mesh 1 without fertilizer, M2WF=Mesh 2 with fertilizer and M2NF=Mesh 2 without fertilizer). Pots were prepared and seeds were sown at a depth of 2 cm. Water was applied after sowing the seeds.

    Fertilizer (N-P-K) was added 2 days after germination (quantities of materials are given in Table 11). Plants were harvested after one month and washed with DI water. Excess water was removed and the total fresh weight was measured.

Amount of material used to prepare pots for test

Effect of biochar in combination with MSWI bottom ash on corn (Zea mays L.)
    Nitrogen, phosphorous and potassium are the three main nutrients required for plant growth (Tucker, 1999). Both MSWI fly ash and bottom ash have been tested previously for their possible application in agriculture (Lam, 2010). As MSWI bottom ash contains tolerable volumes of phosphorous and potassium, it may be used as a partial replacement for commercial fertilizers, however, there are several restrictions for its use. 

     The presence of heavy metals in bottom ash may be toxic to plants and animals; the high content of salts may induce salt stress in plants; the pH value in each type of soil may affect the mobility of different elements and the leaching of heavy metals into ground water may cause environmental concerns. As a result, case-by-case studies have to be performed in this field (Glordano et al., 1983).

Biochars are known to have a highly porous structure and may be effective in the adsorption of heavy metals, especially in aquatic systems (Liu and Zhang, 2009). However, there is a lack of agreement regarding the influence of organic amendments, such as biochars, on metal immobilization in soils (Beesley et al., 2010). Moreover, the application of biochars to contaminated soil systems has not been systematically investigated to any extent (Beesley and Marmiroli, 2011). Therefore, we examined the effect and the interaction of rice husk biochar, bamboo biochar and MSWI bottom ash on the germination and growth of maize plants.

Accumulation of trace element in plant tissue
    In order to evaluate the accumulation of trace metals, water spinach tissue was analyzed.

    Prior to drying, maize plants were decontaminated using deionized water. Afterwards the plant parts were oven dried for 24 hour at 80°C as recommended by Jones Jr. (2001). In order to ensure a degree of uniformity, the dried plant tissue was grounded and sieved with a 20-mesh screen (0.84 m/m).

    Metal content in plant tissue was determined by using the procedures established by Jones Jr. (2001), digestion in a mixture of HNO3 and HCLO4. Dried tissue (0.5 g) of a sample was placed in a 250 ml digestion tube and 2.5 ml of concentrated HNO3 was added, the digestion tubes where then covered with a glass funnel. The mixture was left standing for a night. The covered digestion tube was digested at 80°C for one hour. The digestion cubes where then removed from the hot plate. After cooling, the glass funnels were replaced and 2.5 ml of HCLO4 were added. The solution was heated at 180°C -200°C until fumes of HCLO4 dissipated and the digestion was clear (colorless). Samples were then removed from the heat and cooled down. The interior walls of the tube were washed down with a little distilled water. The tubes were swirled throughout the digestion to keep the wall clean and prevent the loss of the sample. Deionized water was added until obtaining 25 ml of solution. The solution was filtered with Whatman No. 42 filter paper and <0.45 μm Millipore filter paper. Metal content in the extract will be measured using Atomic-Absorption Spectroscopy (AA).

Acid digestion taking place