African Journal of Biotechnology Vol. 10(81), pp.

18775-18780, 16 December, 2011

Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB11.2751
ISSN 1684–5315 © 2011 Academic Journals

Full Length Research Paper

Bioconversion of empty fruit bunches (EFB) and palm

oil mill effluent (POME) into compost using
Trichoderma virens
Dayana Amira, R.1, Roshanida, A. R.1*, Rosli, M. I.1, Siti Fatimah Zahrah, M. F.1, Mohd Anuar,
J.1 and Nazrul Adha, C. M.2
1
Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310, Skudai
Johor, Malaysia.
2
Agrobase Trading Sdn. Bhd., No 7, Tingkat 1, Jalan Bakek, 82000 Pontian, Johor, Malaysia.
Accepted 7 November, 2011

This study shows the performance of Trichoderma virens as an activator for conversion of empty fruit
bunches (EFB) and palm oil mill effluent (POME) into compost. EFB and POME are two abundant
wastes produced by oil palm industries which keep accumulating. Since there is no proper way to
dispose these wastes, a potential way is to turn them into value-added product which is compost.
However, normal composting will take about 4 to 6 months and additional pure fungi on compost can
reduce the time to only 21 to 45 days. It also promotes plant growth and fight plant diseases. T. virens
is one of the potential fungus activator and the enzyme production by this specific fungus has been
studied. Biodegradation of EFB and POME supplemented with T. virens and organic N (chicken manure)
gave significant changes as compared to EFB and POME alone. Application of T. virens resulted in
higher xylanase and cellulase activities which lead to rapid degradation of cellulose and
hemicelluloses. Compost with T. virens has higher xylanase activity on day 36 which is 4.43
mol/(min.mg) as compared to the control which has 3.48 mol/(min.mg). The cellulase activity is
13.214 FPU/mg and 11.314 FPU/mg for compost with T. virens and compost without bioinoculant on day
36, respectively. The N, P, K content of compost with T. virens increased significantly after maturation
which is 1.304, 0.5034 and 0.645%, respectively. This result shows that T. virens played a great role by
shortening the composting period of EFB and POME while producing nutrient-enriched compost.

Key words: Empty fruit bunches (EFB), palm oil mill effluent (POME), bioconversion, Trichoderma virens.

INTRODUCTION

In Malaysia, palm oil industries generate many liquid and could solve several problems including reducing the high
solid wastes, especially the palm oil mill effluent (POME) load of waste produced by palm oil industry. It can also
and empty fruit bunch (EFB) which keeps accumulating. create a market for biofertilizer which have proven to be
If there is no proper way to manage these wastes, it will more effective and cheaper than chemical fertilizer. The
contribute to land pollution and increase the waste industry can generate profit through the waste and the
treatment cost. One potential way to turn the wastes into application of biofertilizer in agriculture would treat the
value-added product is by converting it into compost soil diseases resulting from extensive use of chemical
through microorganism degradation. This conversion fertilizer by returning its biodiversity and fertility.
Conventionally, natural composting takes four to five
months to reach maturity. However, composting process
*Corresponding author. E-mail: [email protected]. can be shortened to three to four weeks only by mixing
the organic material with certain microorganisms.
Abbreviations: EFB, Empty fruit bunch; POME, palm oil mill Composting process can be accelerated to one month by
effluent. inoculation of cellulolytic fungi such as Aspergillus and
18776 Afr. J. Biotechnol.

Table 1. Lignocellulosic content of mixed composition of raw materials with T. virens.

Parameter Percentage reduction Day 36 Total percentage

Day 0 Day 22
(%) until day 22 (%) (After maturation) reduction (%)
Cellulose 52.581 31.26 21.321 26.771 25.81
Hemicellulose 25.456 19.532 5.924 17.897 7.559
Lignin 19.052 10.006 9.046 5.598 13.454
All values are mean of two replicates.

Trichoderma (Biswas and Narayanasamy, 2002). There- thimbles and refluxed for 2 h with 150 ml distilled water at 100°C.
fore, fungi from Trichoderma spp. which has been proven Then, the sample was oven-dried for 24 h. Next, the sample was
refluxed for two more hours in 150 ml H2SO4 at 100°C. After the
to effectively degrade the organic matter into minerals second reflux, the sample was oven-dried for 24 h. The sample was
can be utilized for this purpose. treated with 10 ml of 72% (v/v) H2SO4 at room temperature for 2 h.
Plant growth promoting fungi (PGPF), Trichoderma It was diluted to 0.5 M H2SO4 and was refluxed at 100°C for 2 h.
spp. is believed to confer benefits to plants not only by After the third reflux, the sample was oven-dried and weighed.
promoting the growth but also defending them from
infection (Pandya and Saraf, 2010). Trichoderma virens
Xylanase assay
inoculated in composted chicken manure and rye cover
crops provided significant weed control (Héraux, 2005). Xylanase activity was measured using US Army Natick Research &
There is also another technology which has been Development Laboratories method (Ghose and Bisaria 1987).
developed by Cuevas (1997), and it is the development Enzyme was extracted with citrate buffer of pH 4.8. Then, 1.5 g
of the windrow type of composting, and the main sample of enzyme filtrate was incubated with p-nitrophenyl-beta-D-
modernization is by using pure cultures of Trichoderma xyloparanoside and volume was added to 1.5 ml with 0.05 M citrate
buffer. All tubes were incubated at 50°C for 30 min. Three milliliters
harzianum. Besides, a research done by Gaind and Nain of di-nitrosalicylic acid was added to the sample and then the
(2007) shows that incorporation of paddy straw in soil in sample was boiled in a water bath. Xylanase activity was measured
conjunction with Trichoderma reesei can improve soil in terms of mol xylose produced/min/mg.
biochemical properties. It was also reported by Pandya
and Saraf (2010) that genus Trichoderma is an important
Cellulase assay
biocontrol agent of several soil borne phytopathogens.
Cellulase activity was tested using the method of National
Renewable Energy Laboratory (Adney and Baker, 2008). Enzyme
MATERIALS AND METHODS was first extracted with citrate buffer at pH 4.8. Next, 1.5 g sample
of enzyme filtrate was incubated with filter paper. Citrate buffer
The substrates used in this study were EFB and POME, obtained (0.05 M) was added to the sample to make upto 1.5 ml of the total
from the palm oil plantation of Persatuan Peladang Negeri Johor in volume. All tubes were incubated at 50°C for 30 min. Three
Kahang, Johor. Chicken dung was obtained from animal farm in milliliters of di-nitrosalicylic acid was added and the sample was
Ayer Baloi district, Johor. All raw materials were stored at 4°C for boiled in a water bath. Cellulase activity was measured in terms of
immediate use. filter paper unit per mg (FPU/mg).

Chemical composition of materials RESULTS AND DISCUSSION

The chemical composition of the EFB, POME and chicken dung
(small amount of additional raw material which is the nitrogen Lignocellulosic content
source) were analyzed. Nitrogen, phosphorus and potassium con-
tent were analysed using the Spectroquant Kit by Merck. The lignocellulosic content of mixed composition of EFB,
POME and chicken dung are presented in Tables 1 and 2
for both compost with T. virens and compost without
Inoculation of fungi bioinoculant, respectively. On day 22, the percentage
The fungal strain of T. virens was procured from Universiti
reduction of all three components was significantly high
Kebangsaan Malaysia (UKM). The fungal culture was maintained for compost with T. virens as compared to compost
by subculturing it on potato dextrose agar and keeping it at room without bioinoculants. The reduction of cellulose content
temperature for 7 days. was 21.321 and 2.691% for compost with T. virens and
compost without bioinoculant, respectively. The reduction
of hemicellulose content is 5.924% for compost with T.
Lignocellulosic content
virens and 1.358% and for compost without bioinoculant.
Lignocellulosic content was analysed using Datta’s method (1981) The reduction of lignin content for compost with T. virens
with 3 stages of reflux. One gram of sample was inserted into and compost without bioinoculant is 9.046 and 2.069%,
 Dayana Amira et al. 18777

Table 2. Lignocellulosic content of mixed composition of raw materials without bioinoculants.

Parameter Percentage reduction Day Day Day Day 92 Total percentage

Day 0 Day 22
(%) until day 22 (%) 43 64 85 (after maturation) reduction (%)
Cellulose 52.581 49.89 2.691 42.756 32.875 28.907 25.075 23.674
Hemicellulose 25.456 24.098 1.358 24.578 20.065 18.08 17.964 7.376
Lignin 19.052 16.983 2.069 14.002 12.85 9.006 7.502 10.046
All values are mean of two replicates.
Activity {µmol/(min.mg)}

Time (day)

Figure 1. Xylanase activity versus time of composting with T. virens and composting without bioinoculant.

respectively. High percentage in compost with T. virens Enzyme assay

indicates that the component is efficiently degraded. This
finding is similar to that of Singh and Sharma (2002) who Enzyme assay is one of the characteristics that respond
reported a rapid degradation of wheat straw with the use more quickly as compared to organic matter composition
of fungi as compared to compost without bioinoculant in the compost. Incorporation of EFB, POME and chicken
which has slower degradation. manure with inoculated fungi influences the enzyme
After maturation on day 36, the total percentage reduc- activities in compost. Figure 1 shows the xylanase activity
tion of lignocellulosic for compost with T. virens is 25.81, versus time of compost without bioinoculant (control) and
7.559 and 13.454% for cellulose, hemicellulose and compost with T. virens. It shows that both composts have
lignin, respectively. However, the compost without bioino- increasing xylanase activity. However, compost with T.
culant takes up to 92 days to reach maturity with the total virens has higher activity with the increment of
percentage reductions of lignocellulosic at 23.674, 7.376 27.32609% on day 8 and up to 39.97576% on day 36.
and 10.046% for cellulose, hemicellulose and lignin, This is due to the existence of T. virens which produces
respectively. It was observed that all three organic xylanase. Compost without bioinoculant only approaches
components of cellulose, hemicelluloses and lignin 12.21415% increment on day 8 and increased to
decreased significantly for both composts. However, the 23.61561% on day 36. Highest xylanase activity for
compost with T. virens matured on day 36, while compost compost without bioinoculant was 3.48 mol/(min.mg)
without bioinoculant matured on day 92. This shows that which is on day 36. The highest cellulase activity is for
T. virens reduced the maturation time by 60.9% as compost mixture with T. virens on day 36 which is 4.32
compared to compost without bioinoculant. Therefore, mol/(min.mg). Trichoderma spp. is definitely known to
addition of T. virens as accelerating agent affects the produce enzyme with high xylanolytic activity (Wong and
speed of composting and maturation time (Haddadin et Saddler, 2002).
al., 2009). Figure 2 shows the cellulase activity versus time of
18778 Afr. J. Biotechnol.

Activity (FPU/mg)

Time (day)
Figure 2: Cellulase activity versus time of composting with T. virens and composting without
bioinoculant.

compost without bioinoculant (control) and compost with content as compared to compost without bioinoculant.
T. virens. It shows that both composts have increasing High total nitrogen content of compost with T. virens
cellulase activity. However, it is obvious that T. virens might be due to enhanced decomposition of organic
have higher activity for the total of 36 days of composting. matter by the fungi (Pramanik et al., 2007). High total
The increase of compost with T. virens is 4.94% on day 8 nitrogen is also governed by the initial nitrogen content of
and up to 18.61% on day 36. When compared with the raw materials. Besides, the increasing concentration
compost without bioinoculant, the increase was observed on nitrate-nitrogen (NO3-N) at the end of the composting
to be 0.29% on day 8 and up to 4.94% on day 36. The process increases the total nitrogen content. However,
highest xylanase activity for compost without bioinoculant ammonium-nitrogen (NH4-N) is high in the early stage of
is 13.214 FPU/mg which is on day 36. The highest composting but decrease gradually during maturation of
activity of cellulase is for compost with T. virens which is the compost (Young et al., 2005).
11.314 FPU/mg on day 36. Phosphorus content is also higher with regards to
Both xylanase and cellulase activity of compost with T. maturation for both compost and this is because
virens show increasing trend towards the maturity of phosphorus is not lost by volatilization or lixiviation during
compost and this is in agreement with Gaind and Nain the composting process. The concentration of phosphor-
(2007) which used Aspergillus awamori and T. reesei. rus increases as composting proceeds (Young et al.,
Compost with T. virens has high enzyme activities for 2005). Similarly, potassium is high for both compost as
-
both xylanase and cellulase which indicates that the compared to initial value since PO4 ions from humic
compost was effectively degraded. This is due to the colloids are released into the system (Pramanik et al.,
existence of fungal which can produce digestive enzyme 2007). For compost with T. virens, there was higher
such as xylanase and cellulose. percentage of phosphorus and potassium due to the
presence of fungal which plays an important role in
increasing the P and K content during the process.
Macronutrients content Table 5 shows the N, P, K percentage of compost from
different raw materials and microorganism studied by
Tables 3 and 4 show the percentage of nitrogen, different researchers. The nitrogen percentage in this
phosphorus and potassium content of both composts at study (1.304%) was observed to be in the range of other
before and after maturation. Chemical analysis shows studies which is 0.98 to 2%. Phosphorus and potassium
that compost with T. virens has higher macronutrients percentage in this study is observed to be lower as com-
 Dayana Amira et al. 18779

Table 3. Composition of compost at initial stage.

Parameter N (%) P (%) K (%)

Compost (EFB, POME and chicken dung) 0.913 0.370 0.603

Table 4. Composition of the compost.

Parameter N (%) P (%) K (%)

Compost with T. virens (day 36) 1.304 0.543 0.645
Compost without bioinoculant (day 92) 0.961 0.412 0.633

Table 5. Nitrogen, phosphorus and potassium content in the compost.

Materials/microorganism N (%) P (%) K (%) Reference

Wheat straw + T. harzianum + A. niger + Azotobacter chroococcum 0.98 0.19 0.55 Singh and Sharma (2002)
Olive pomace + T. harzianum + Phanerochaete chrysosporium 1.6 1.1 19.8 Haddadin et al. (2009)
Rice straw + weeds + chicken manure + T. harzianum 2.0 2.9 1.9 Virginia (1997)
Rice straw + okara + vinasse + buffalo manure + rock phosphate 1.94 1.95 0.953 Rashad et al. (2010)
empty fruit bunches + palm oil mill effluent + chicken dung + T. virens 1.304 0.543 0.645 This study
Weight (g)

Time (day)
Figure 3. Microbial biomass of compost with T. virens and compost without bioinoculant.

compared to most of the other studies, which might be virens and compost without bioinoculant. An increasing
due to several factors such as composition and type of number of T. virens cell was observed during the
raw materials and mixed microorganism. Therefore, an composting period. T. virens spread and grow vigorously
optimization study will be further carried out in order to by producing many cells in order to secrete digestive
improve the percentage of N, P and K. enzyme and degrade the lignocellulosic component. The
increasing trend of the cell is parallel with the increase of
the enzyme activity, hence will also make it to be a good
Microbial biomass biocontrol agent when it is applied to soil. Trichoderma
spp. are high colonizers of their habitat and easily utilizes
Figure 3 shows the microbial biomass of compost with T. the substrate and secreting digestive enzymes.
18780 Afr. J. Biotechnol.

Conclusion Héraux Farah MG, Hallett Steven G, Weller Stephen C (2005).

Combining Trichoderma virens-inoculated compost and a rye cover
crop for weed control in transplanted vegetables. Biol. Control. pp.
Composting of EFB and POME with T. virens shortened 3421-3426.
the composting period to 60.9% as compared to compost Pandya U, Saraf M (2010). Application of Fungi as a Biocontrol Agent
without bioinoculant. Xylanase and cellulase production and their Biofertilizer Potential in Agriculture. J. Adv. Dev. Res. 1(1):
90-99.
was observed to be high in compost with T. virens hence,
Pramanik P, Ghosh GK, Ghosal PK, Banik P (2007). Changes in
lead to rapid degradation of both hemicelluloses and organic – C, N, P and K and enzyme activities in vermicompost of
cellulose, respectively. T. virens also contributes towards biodegradable organic wastes under liming and microbial inoculants.
the higher percentage of nitrogen, phosphorus and pota- Bioresour. Technol. 98: 2485-2494.
Rashad Ferial M, Saleh Walid D, Moselhy Mohamed A (2010).
ssium content. The outcome of this study is beneficial in Bioconversion of rice straw and certain agro-industrial wastes to
converting the waste into value-added products in a short amendments for organic farming systems: 1. Composting, quality,
time with additional good impacts on the plant as well. stability and maturity indices. Bioresour. Technol. 101: 5952-5960.
Application of compost with T. virens to plant is expected Singh A, Sharma S (2002). “Composting of a Crop Residue Through
Treatment With Microorganisms and Subsequent Vermicomposting”.
to have benefits and therefore thorough studies should
Bioresource. Technol. 85: 107-111.
be done further. Wong Ken KY, Saddler John N (1992). Trichoderma Xylanase, Their
Properties and Application. Biotechnology. 12(56): 413-435.
Young CC, Rekha PD, Arun AB (2005). What Happens During
Composting? Food and Fertilizer technology Centre for The Asian
ACKNOWLEDGEMENT
and Pacific Region. pp. 01-12.

The authors thank Universiti Teknologi Malaysia and

Agrobase Trading Sdn. Bhd. for the financial and
technical support of this research.

REFERENCES

Adney B, Baker J (2008). Measurement of Cellulase Activities. Nat.

Renewable. Energy Lab. Issue Date: 08/12/1996. pp. 1-8.
Biswas DR, Narayanasamy G (2002). Mobilization of phosphorus from
rock phosphate through composting using crop residue-
fertilizer. News B. pp. 53-56.
Datta R (1981). Acidogenic Fermentation of Lignocellulosic Acid Yield
and Conversion of Components. Biotechnol. Lett. 161(12): 1317-
1322.
Gaind S, Nain L (2007). Chemical and biological properties of wheat soil
in response to paddy straw incorporation and its biodegradation by
fungal inoculants. Biodegradation. 18: 495-503.
Ghose TK, Bisaria VS (1987). Measurement of Hemicellulase Activities
Part 1: Xylanases. Pure. Appl. Chem. 59(12): 1739-1752.
Haddadin Malik SY, Haddadin J, Arabiyat Omar I, Hattar B (2009).
Biological Conversion of Olive Pomace into Compost by Using
Trichoderma harzianum and Phanerochaete chrysosporium.
Bioresour. Technol. 100: 4773-4782.