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Use of Jatropha curcas hull biomass for bioactive compost production

D.K. Sharmab, A.K. Pandeya, Lataa,
a b

Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India Division of Environmental Sciences, Indian Agricultural Research Institute, New Delhi 110012, India

art i cle info

Article history: Received 21 July 2006 Received in revised form 3 July 2007 Accepted 2 May 2008 Keywords: Jatropha hulls Composting Lignocellulolytic fungi Phytotoxicity

ab st rac t
The paper deals with utilization of biomass of Jatropha hulls for production of bioactive compost. In the process of Jatropha oil extraction, a large amount of hull waste is generated. It has been found that the direct incorporation of hull into soil is considerably inefcient in providing value addition to soil due to its unfavorable physicochemical characteristics (high pH, EC and phenolic content). An alternative to this problem is the bioconversion of Jatropha hulls using effective lignocellulolytic fungal consortium, which can reduce the phytotoxicity of the degraded material. Inoculation with the fungal consortium resulted in better compost of jatropha hulls within 1 month, but it takes nearly 4 months for complete compost maturation as evident from the results of phytotoxicity test. Such compost can be applied to the acidic soil as a remedial organic manure to help maintaining sustainability of the agro-ecosystem. Likewise, high levels of cellulolytic enzymes observed during bioconversion indicate possible use of fungi for ethanol production from fermentation of hulls. & 2008 Published by Elsevier Ltd.

1.

Introduction

Due to severe energy crisis and escalation of petroleum prices, alternate energy sources are gaining importance. The concept of substituting biodiesel produced from plantations of Jatropha on eroded soils for conventional diesel fuel has gained widespread attention in India. Jatropha curcas is being grown in 0.4 mha of marginal lands covering forest and nonforest lands in various states across the country. Extraction of oil from jatropha seeds generate substantial amount of hulls waste. One tonne of jatropha seeds will provide about 350-l oil and 2.40 tonne hulls. Therefore, in future, disposal of jatropha hulls will create problem if Jatropha is being used at a commercial level for biodiesel production. Because the hulls have low density, it is not of economic interest to transport
Corresponding author. Tel.: +91 11 25847649; fax: +91 11 25846420.

them over long distances for processing. Finding a lower cost, environmentally sustainable, long-term solution for handling Jatropha hulls is therefore of critical importance. Keeping in view the entire problem, this study was undertaken to prepare bioactive compost from jatropha hulls through consortium of lignocellulolytic fungi. This nutrient-enriched compost may be used further as manure for organic farming.

2.
2.1.

Materials and methods

Compost accelerators

Four mesophilic strains of Aspergillus nidulans ITCC 2011, Trichoderma viride ITCC 2211, Phanerochaete chrysosporium NCIM

E-mail address: latarajat@yahoo.co.in ( Lata). 0961-9534/$ - see front matter & 2008 Published by Elsevier Ltd. doi:10.1016/j.biombioe.2008.05.002

Please cite this article as: Sharma DK, Pandey AK, Lata. Use of Jatropha curcas hull biomass for bioactive compost production. Biomass and Bioenergy (2008), doi:10.1016/j.biombioe.2008.05.002

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1073 and Aspergillus awamori F-18, screened on the basis of hyper cellulase and xylanase activity, were used as inoculum to accelerate the composting process [1].

3.

Results and discussion

2.2.

Inoculum development

Inoculum for all the four cultures were produced on boiled sorghum seeds supplemented with 2% calcium carbonate and 4% calcium sulfate. Cultures were incubated at 30 1C for 15 days, and these grains with mycelium and spores were used as inoculum. All the four cultures were mixed in equal quantity to obtain the mixed inoculum and applied at 300 g t1 of substrate.

2.3.

Composting piles

Jatropha fruit shell was used as substrate for composting without grinding. Forty kg substrate was lled in perforated cemented pits of size 1 m3 with 100% moisture in the beginning. Mixed fungal inoculum was applied at 300 g t1 substrate, keeping an uninoculated control. The piles were periodically aerated by turning the contents. Water was added during fortnightly turnings to maintain the moisture at 60%.

2.4.

Sampling and analyses

Composite samples of 500 g were taken from each pit. A portion of it was air dried, homogenized and nally ground to pass through a 1 mm sieve. Total organic matter, organic carbon, total Kjeldahls nitrogen, C/N, fungal biomass [2], humus [3], pH and EC were measured in these samples. Total water extractable and pyrophosphate extractable carbon were estimated by the chromate oxidation method [4]. Microbial activity was measured in terms of uorescein diacetate (FDA) hydrolysis at 30 1C by incubating the sample for 2 h and measuring the absorbance at a wavelength of 490 nm [5]. Another portion of sample was stored in a refrigerator at 8 1C for enzyme assay. All the sample analyses were carried out in triplicate.

2.5.

Enzyme assay

The composition of substrate (Jatropha hulls) used for the compost preparation is presented in Table 1. Jatropha hull, due to low nitrogen content, had a wide C/N ratio of about 66.93, which is higher than that recommended (3545) for composting. In the present study, C/N was not lowered in the beginning of composting as Line [14] had reported that an initial ratio of 60 was the most benecial ratio for composting pulp and paper by-products. The electrical conductivity of the substrate was 7.50, an indication of high salt concentration. Likewise, pH of substrate was recorded to be 8.1. Jatropha hull was also found to be rich in phenolics. Similar composition has been reported by Gubitz et al. [15] for J. curcas hulls. Table 2 presents the average values of physiological parameters analyzed in the degraded hulls after composting for 1 month. The C/N ratio (1216) indicates the suitable level of decomposition in compost. A range of 1215 is reported in mature compost by Golueke [16]. The lower C/N ratio obtained in this work is explained by the fact that the amount of carbon is reduced by way of partial conversion to CO2, while nitrogen continues to be recycled. Although the initial C/N ratio of hulls was high (66:1), degradation completed at a faster rate, and lower C/N ratio was achieved within 30 days. This may be possibly due to the presence of simple compounds in the substrate, which might have resulted in faster colonization by microbes. Likewise, degradation was more pronounced in the piles inoculated with compost accelerators. The degraded hulls had more EC (E10) as compared to raw substrate. Electrical conductivity measures the amount of soluble salts in the compost sample and most desired values range from 3 to 5. The values lower than these indicate the lack of available minerals, while the values higher than this will inhibit the biological activities. Likewise, pH of compost increased up to 10 after decomposition. Increase in pH may be due to the production of NH+ during proteolysis and when O2 4 is not limiting, organic acid production will be low but NH3 emission will be high, hence pH of compost rises [17]. Similarly, increase in EC can be correlated with water evaporation as well as due to concentration effect. Therefore,

The assay of various enzyme activities was based on the release of products and its quantitative determination in a reaction mixture. The dehydrogenase and alkaline phosphatase were estimated by the method of Casida et al. [6] and Tabatabai and Bremner [7] using triphenyl tetrazolium chloride and p-nitrophenyl as substrate, respectively. Cellulase and xylanase activities were performed on samples of aqueous compost extracts by using lter paper and xylan as substrate by the standard methods of Ghose and Bailey et al. [8,9], respectively. The amount of reducing sugar released was estimated by the dinitrosalicylic acid method [10]. Total phenol and soluble protein concentration were analyzed in the aqueous extract of compost by Bray and Thorpe and Lowry et al. [11,12], respectively. Phytotoxicity was evaluated by means of the seed germination test using cress seeds (Lepidium sativum) at 27 1C with aqueous extract of compost [13].

Table 1 Chemical composition of Jatropha fruit hulls (eshy mesocarp) Parameters

Dry matter (%) Carbon (%) Crude protein (%) Nitrogen (%) C/N Available phosphorus (mg g1) pH EC Soluble protein (mg g1) Total soluble phenolics (mg g1)

Values
89.8 46.05 4.34.5 0.688 66.93 145.983 8.1 7.50 0.762 1.831

Please cite this article as: Sharma DK, Pandey AK, Lata. Use of Jatropha curcas hull biomass for bioactive compost production. Biomass and Bioenergy (2008), doi:10.1016/j.biombioe.2008.05.002

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Table 2 Physiochemical and biochemical properties of Jatropha curcas hulls and extracellular enzymes in inoculated and uninoculated compost Parameters Uninoculated compost Inoculated composta

Physiochemical parameters Organic carbon (%) Total nitrogen (%) C/N Available nitrogen (%) Total phosphorus (%) Available phosphorus (mg g1) Total K (%) Total Na pH EC Humic substances (%) Total pyrophosphate extractable C (%) Total water extractable C (%) Biochemical parameters Total soluble protein (mg g1) Total extractable phenolics (mg g1) Microbial activity Fungal biomass (mg Nacetyl glucosamine g1) FDA hydrolysis (mg uorescein g1 h1) Dehydrogenase (mg TPF g1 day1) Alkaline phosphatase (mg pNP day1) Extracellular enzymes FPase (IU g1) CMCase (IU g1) Cellobiase (IU g1) Xylanase (IU g1) Lignin peroxidase (IU g1) Laccase (IU g1)
a

23.405 1.38 16.96 0.03 0.40 290.752 7.15 5.3 10.20 10.90 14 0.089 0.061

17.97 1.46 12.30 0.04 0.21 249.222 6.30 4.8 10.25 10.15 15 0.102 0.074

1.461 5.998

1.317 6.116

6.32 23.2 234.40 748.49

6.49 31.4 330.26 616.69

uninoculated compost. Total extractable phenol concentration increased approximately 5-fold in compost as compared to undegraded hulls. This indicates liberation of phenolic monomers from the lignin component of jatropha shells by microora active during decomposition. However, microbial activity in terms of FDA hydrolysis, dehydrogenase and fungal biomass in terms of N-acetyl-glucosamine, was more in compost inoculated with fungi. Smith and Hughes [20] had reported a uorescein production rate of 4 mg g1 h1 during composting of garden refuse. The abundance of available C (0.0610.074%) might have resulted in higher uorescein liberation rate observed in the present investigation. Dehydrogenase has been considered an indicator of overall microbial activity because it occurs intracellularly in all living microbial cells, and is linked with the microbial respiratory process. This parameter is also adjudged to be the most suitable indicator of compost maturity [21]. Alkaline phosphatase activity is inversely related with available P in compost [22]. In this study less activity (616.69 mg PNP day1) is observed in inoculated compost. Similar results were reported by Saavedra et al. [23] in olive mill cake composting. Since combination of four fungi was used as inoculum during composting fungal biomass in terms of N-acetyl-glucosamine (6.49 mg g1) was higher in inoculated compost. In general, activity of lignocellulolytic enzyme was more in inoculated compost. Most pronounced effect was observed in cellobiase, the rate-limiting enzyme during cellulolysis. Likewise, there was more expression of laccase (1.67 IU g1) in compost inoculated with fungi. This may be due to the presence of white rot lignolytic fungus P. chrysosporium in the inoculum. Several workers have also reported stimulation of composting/ biodegradation of different agroresidues by inoculation with mixture of saprobic fungi [24], lignocellulolytic fungi [25].

0.232 2.136 0.010 0.293 1.33 0.40

0.398 2.414 0.060 0.279 1.53 1.67

4.

Conclusion

Inoculated with mixture of four lignocellulolytic fungi.

a high pH and EC value is a point of concern if jatropha compost has to be used as manure in the eld. During the composting process, the organic fractions are partially degraded by aerobic microorganisms and the starting material is transformed through a variety of biological and biochemical processes in which enzymes play a role [16,18]. Microbes in the compost pile cannot directly metabolize the insoluble particles of organic matter. Therefore, they produce hydrolytic extracellular enzymes to depolymerize the larger compounds to smaller fragments, which can be assimilated by microorganisms in compost [19]. Since all the hydrolytic enzymes participating in decomposition of macromolecules (lignocellulose) are extracellular in nature, the total soluble protein content was estimated in the compost extract. No signicant differences were observed in the inoculated and

Our results conrmed that inoculation of lignocellulolytic fungi resulted in better compost of jatropha hulls within 1 month. However phytotoxic compounds were present in the compost, resulting in the low germination of L. sativum. When this compost matured for 4 months, phytotoxicity reduced in terms of the germination index (E80%). Therefore, it would be advisable that composting may be continued for 4 months to reduce the phytotoxicity of compost by action of enzymes secreted by lignocellulolytic fungi. Since, compost has alkaline pH it can be applied to acidic soil as manure to neutralize soil pH. The potential of lignocellulolytic fungi, used in this study, to produce higher quantities of cellulolytic enzymes can be tapped in an effective manner by using them for SSF or SmF of jatropha hulls. The resultant crude enzyme preparations can be exploited for fermentation of hulls to produce ethanol that can be used as additional source of biofuel.
R E F E R E N C E S

[1] Lata, Gaind S, Pandey AK. Chemical characterization of composts prepared with diversied agro wastes. Indian Journal of Microbiology 2005;45(3):2457.

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[2] Aidoo KE, Hendry R, Wood BJB. Estimation of fungal growth in solid state fermentation systems. European Journal of Applied Biotechnology 1981;12:69. [3] Kononova MN. Soil organic matter, its nature, its role in formation of soil fertility. Oxford: Pergamon Press; 1966. [4] Jackson ML. Soil chemical analysis. New Delhi: Prentice Hall of India Pvt. Ltd.; 1967. [5] Swisher R, Carroll GC. Fluroscein diacetate as an estimator of microbial biomass on coniferous needle surface. Microbial Ecology 1980;6:21726. [6] Casida LE, Klein DA, Santoro T. Soil dehydrogenase activity. Soil Science 1964;98:3716. [7] Tabatabai MA, Bremner JM. Use of p-nitrophenol phosphate in assay of soil phosphatase activity. Soil Biology & Biochemistry 1969;1:3017. [8] Ghose TK. Measurement of cellulase activities. Pure & Applied Chemistry 1987;59:25768. [9] Bailey MJ, Biely P, Poutanen K. Interlaboratory testing of method for assay of xylanase activity. Journal of Biotechnology 1992;23:25770. [10] Miller GL. Use of DNSA reagent for determination of reducing sugar. Analytical Chemistry 1959;31:4268. [11] Bray HC, Thorpe WV. Analysis of phenolic compounds of interests in metabolism. Methods in Biochemical Analysis 1954;1:2732. [12] Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. Journal of Biological Chemistry 1951;193:26577. [13] Zucconi F, Forte M, Monaco ADE, Bertoldi M. Biological evaluation of compost maturity. Biocycle 1981;22:279. [14] Line MA. Compost recycling of wood bre waste produced by paper manufacture. Compost Science and Utilization 1995;3(1):3945. [15] Gubitz GM, Mittelbach M, Trabi M. Exploitation of the tropical oil seed plant Jatropha curcas L. Bioresource Technology 1999;67:7382.

[16] Golueke CG. Bacteriology of composting. Biocycle 1992;33:557. [17] Paredes C, Roig A, Bernal MP, Sanchez-Monedero MA, Cegarra J. Evolution of organic matter and nitrogen during cocomposting of olive mill wastewater with solid organic wastes. Biology and Fertility of Soils 2000;32:2227. [18] Garcia C, Hernandez T, Costa F, Ceccanti B, Ciardi C. Changes in ATP content, enzyme activity and inorganic nitrogen species during composting of organic wastes. Canadian Journal of Soil Science 1992;72:24353. [19] Godden B, Penninckx M, Pierard A, Lannoye R. Evolution of enzyme activities and microbial populations during composting of cattle manure. European Journal of Applied Microbiology and Biotechnology 1983;17:30610. [20] Smith DC, Hughes JC. Changes in maturity indicators during the degradation of organic wastes subjected to simple composting procedures. Biology and Fertility of Soils 2004; 39:2806. [21] Gaind S, Pandey AK, Lata. Biodegradation study of crop residues as affected by exogenous inorganic nitrogen and fungal inoculants. Journal of Basic Microbiology 2005;45(4): 30111. [22] Burns RG. Soil enzymes. New York: Academic Press; 1978. [23] Saavedra M, Benitez E, Cifuentes C, Nogales R. Enzyme activities and chemical changes in wet olive cake after treatment with Pleurotus ostreatus or Eisenia fetida. Biodegradation 2006;17:93102. [24] Sampedro I, Aranda E, Martin J, Garcia-Garrido JM, GarciaRomera I, Ocampo JA. Saprobic fungi decrease plant toxicity caused by olive mill residues. Applied Soil Ecology 2004;26:14956. [25] Lu W-J, Wang H-T, Nie Y-F, Wang Z-C, Huang D-Y, Qiu X-Y, et al. Effect of inoculating ower stalks and vegetable waste with ligno-cellulolytic microorganisms on the composting process. Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes 2004;39(56):87187.

Please cite this article as: Sharma DK, Pandey AK, Lata. Use of Jatropha curcas hull biomass for bioactive compost production. Biomass and Bioenergy (2008), doi:10.1016/j.biombioe.2008.05.002