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Short communication

Use of Jatropha curcas hull biomass for bioactivecompost production

D.K. Sharmab, A.K. Pandeya, Lataa,�

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

a r t i c l e i n f o

Article history:

Received 21 July 2006

Received in revised form

3 July 2007

Accepted 2 May 2008

Available online 17 June 2008

Keywords:

Jatropha hulls

Composting

Lignocellulolytic fungi

Phytotoxicity

nt matter & 2008 Elsevieioe.2008.05.002

thor. Tel.: +91 11 25847649latarajat@yahoo.co.in ( La

a b s t r a c 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

inefficient 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 Elsevier Ltd. All rights reserved.

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 non-

forest 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

r Ltd. All rights reserved.

; fax: +91 11 25846420.ta).

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. Materials and methods

2.1. Compost accelerators

Four mesophilic strains of Aspergillus nidulans ITCC 2011,

Trichoderma viride ITCC 2211, Phanerochaete chrysosporium NCIM

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Table 1 – Chemical composition of Jatropha fruit hulls(fleshy mesocarp)

Parameters Values

Dry matter (%) 89.8

Carbon (%) 46.05

Crude protein (%) 4.3–4.5

Nitrogen (%) 0.688

C/N 66.93

Available phosphorus (mg g�1) 145.983

pH 8.1

EC 7.50

Soluble protein (mg g�1) 0.762

Total soluble phenolics (mg g�1) 1.831

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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].

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 t�1 of substrate.

2.3. Composting piles

Jatropha fruit shell was used as substrate for composting

without grinding. Forty kg substrate was filled in perforated

cemented pits of size 1 m3 with 100% moisture in the

beginning. Mixed fungal inoculum was applied at 300 g t�1

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 finally ground to

pass through a 1 mm sieve. Total organic matter, organic

carbon, total Kjeldahl’s 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 fluorescein 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 assay of various enzyme activities was based on the

release of products and its quantitative determination in a

reaction mixture. The dehydrogenase and alkaline phospha-

tase 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 filter 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 germina-

tion test using cress seeds (Lepidium sativum) at 27 1C with

aqueous extract of compost [13].

3. Results and discussion

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 (35–45) 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 beneficial 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 (12–16) indicates the suitable level

of decomposition in compost. A range of 12–15 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 NH4+ during proteolysis and when O2

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,

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Table 2 – Physiochemical and biochemical properties ofJatropha curcas hulls and extracellular enzymes ininoculated and uninoculated compost

Parameters Uninoculatedcompost

Inoculatedcomposta

Physiochemical parameters

Organic carbon (%) 23.405 17.97

Total nitrogen (%) 1.38 1.46

C/N 16.96 12.30

Available nitrogen (%) 0.03 0.04

Total phosphorus (%) 0.40 0.21

Available phosphorus

(mg g�1)

290.752 249.222

Total K (%) 7.15 6.30

Total Na 5.3 4.8

pH 10.20 10.25

EC 10.90 10.15

Humic substances (%) 14 15

Total pyrophosphate

extractable C (%)

0.089 0.102

Total water extractable C (%) 0.061 0.074

Biochemical parameters

Total soluble protein

(mg g�1)

1.461 1.317

Total extractable phenolics

(mg g�1)

5.998 6.116

Microbial activity

Fungal biomass (mg N-

acetyl glucosamine g�1)

6.32 6.49

FDA hydrolysis (mg

fluorescein g�1 h�1)

23.2 31.4

Dehydrogenase

(mg TPF g�1 day�1)

234.40 330.26

Alkaline phosphatase

(mg pNP day�1)

748.49 616.69

Extracellular enzymes

FPase (IU g�1) 0.232 0.398

CMCase (IU g�1) 2.136 2.414

Cellobiase (IU g�1) 0.010 0.060

Xylanase (IU g�1) 0.293 0.279

Lignin peroxidase (IU g�1) 1.33 1.53

Laccase (IU g�1) 0.40 1.67

a Inoculated with mixture of four lignocellulolytic fungi.

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a high pH and EC value is a point of concern if jatropha

compost has to be used as manure in the field.

During the composting process, the organic fractions are

partially degraded by aerobic microorganisms and the start-

ing 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

significant differences were observed in the inoculated and

uninoculated compost. Total extractable phenol concentra-

tion 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

microflora 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 fluorescein production rate of 4 mg g�1 h�1 during

composting of garden refuse. The abundance of available C

(0.061–0.074%) might have resulted in higher fluorescein

liberation rate observed in the present investigation. Dehy-

drogenase 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 phos-

phatase activity is inversely related with available P in

compost [22]. In this study less activity (616.69mg PNP day�1)

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 g�1) 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. Like-

wise, there was more expression of laccase (1.67 IU g�1) 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].

4. Conclusion

Our results confirmed 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 alka-

line 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 prepara-

tions can be exploited for fermentation of hulls to produce

ethanol that can be used as additional source of biofuel.

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