Malaysias oil palm industry has grown rapidly over the last four decades and it becomes very important agriculture-based industry. Since, then the country has maintained her position as the world leading palm oil producing country. Malaysia is blessed with favourable weather condition, which prevails throughout the year and advantageous for oil palm plantation. Nevertheless, the industry has also generated vast quantity of palm biomass, mainly from milling and crushing palm kernel. The main by-product and waste are Empty Fruit Bunches (EFB), Palm Oil Mill Effluent (POME), palm fibre and palm kernel shell.
Oil Palm Empty Fruit Bunches (OPEFB) as a lignocellulosic residue has potential
as a cheap renewable feedstock for large scale production of bioproduct. Each
year, more than 15 million tonnes of OPEFB were generated by palm oil industry
in Malaysia (Rahman et al., 2007).
Bioconversion of lignocellulosic to bioethanol employs three major steps which
include, pretreatment of OPEFB for breakdown lignin and open up crystalline
structure of cellulosic materials, cellulosic hydrolysis using combination of
enzymes for fermentable sugar (polyoses) production and bioconversion of fermentable
sugar (polyoses) produced to bioethanol. Cellulose degradation by enzymatic
hydrolysis is a reaction carried out by cellulase enzymes, which are very specific.
Three major groups of cellulase are involved in the hydrolysis process of cellulose
(Cao and Tan, 2002) and these are:
||β-1-4-endoglucanase, which attack low crystalline region
in the cellulose fiber creating free chain ends
||β-1-4exoglucanase or cellobiohydrolase, which degrades
the molecule further by removing cellobiose units from the free chain ends
||β-glucosidase or cellobiase, which hydrolyzes cellobiose
to produce glucose
In the pretreatment of OPEFB, physicochemical technologies such as steam explosion,
dilute acid, alkali and oxidant or their combination are several existing pretreatment
method have mostly been developed by Moiser et al.
(2005). However, physical and chemical pretreatment involve high energy
(electricity and steam) and corrosion-resistant, high pressure reactors, which
raise the cost of pretreatment operations and equipment usage (Shi
et al., 2009a). In addition, chemical pretreatment can be unfavourable
to successive of enzymatic hydrolysis and microbial fermentation apart from
producing acidic or alkaline waste water which needs predisposal treatment for
environmental safety (Keller et al., 2003).
Microbial pretreatment which employs microorganism especially fungi and their
enzymes system for degradation of lignin present in the lignocellulosic biomass
is a gentle substitute to harsh chemicals pretreatment. Fungal pretreatment
has been formerly explored to improve cellulosic materials for feed and paper
applications (Hadar et al., 1993). Recently,
this environmental friendly approach has received renewed attention as a pretreatment
technique for enhancing enzymatic saccharification and fermentation of lignocellulosic
biomass to bioethanol (Camarero et al., 1994).
The white-rot basidiomycetes fungus, has attract an interest since it efficiently
degrade lignin. Microbial pretreatment has potential advantages over the existing
physicochemical pretreatment technologies due to reduced energy and material
cost, relatively simple equipment and biological catalyst utilization (Keller
et al., 2003). Though, the practicability of microbial pretreatment
is still questioned essentially due to the really long duration of pretreatment
as well as the complexity in selective degrading of lignin (Hatakka,
1983). Phanerochaete chrysosporium has high growth rate compared
to many other basidiomycetes, exceptional oxidation potential and efficiency
for lignin biodegradation (Singh and Chen, 2008). Phanerochaete
chrysosporium delignification abilities could be enhanced by optimizing
the media and cultivation methods to increase ligninolytic enzyme production
(Reddy and DSouza, 1994).
The objective of this study was to evaluate the lignin content of OPEFB by using kappa number after chemical pretreatment using dilute sodium hydroxide (NaOH) as compared with fungal pretreatment using Phanerochaete chrysosporium ATCC 32629 as model microorganism by liquid solid culture techniques. The production of lignin peroxidase in liquid and solid state fermentation was compared.
MATERIALS AND METHODS
Laboratory Scale Fermentation
This study was conducted in laboratory scale at Institute of Bioscience
and Biomass Technology Center, Universiti Putra Malaysia and SIRIM Berhad, Malaysia,
from October 2008 to April 2009.
The biomass used in this study is shredded OPEFB. It was obtained from Sri
Ulu Langat Palm Oil Mill at Dengkil, Selangor, Malaysia. The raw substrate was
kept in the cold room at 4°C to avoid contamination. The OPEFB was soaked
in detergent for overnight to removed any residual oil and then sun-dried. The
OPEFB was ground by a hammer mill and kept in a dry place.
Organism and Cultivation Condition
Phanerochaete chrysosporium ATCC 32629 was purchased from American
Type Culture Collection. Potato Dextrose Agar (Difco) was used for strain maintenance
Spore production in slant required 5 days of growth at 39°C. Spores
(conidia) are prepared by suspension in sterile water followed by passage through
sterile glass wool. Spore concentration is determined by using spore count method
(5x106 spores mL-1). Two days old vegetative inoculums
cultured in Potato Dextrose Broth was used for both liquid fermentation and
solid state fermentation.
Cultivation ModeSolid State Fermentation (SSF) was carried out in 250
mL Erlenmeyer flask. Two grams of untreated OPEFB was wetted with 8.45 mL media,
setting the moisture content to approximate 80% (wet basis). The composition
for lignin peroxidase and manganese peroxidase production was based on modified
Basal III media (Tien and Kirk, 1988) supplemented with
untreated OPEFB. Fermentation was done at temperature 39°C.
For submerged liquid fermentation, 100 mL media and 2 g of untreated OPEFB was used. Inocula size was fixed at 2% (v/v) using 2 days vegetative inoculum. Media composition and Erlenmeyer flask size were similar to SSF set up.
The OPEFB was delignified using NaOH. Two grams of untreated OPEFB was mixed
together with 200 mL of 2% (w/v) NaOH. The mixture was heated at 90°C for
1, 2 and 3 h, respectively.
Lignin Content Analysis
The kappa number analysis and Klason lignin determination was used to measure
the extent of delignification (TAPPI, 1999). Kappa number
based on potassium permanganate solution consumed by a moisture-free pulp under
specific conditions. The percentage of Klason lignin approximately was determining
based on Technical Association of the Pulp and Paper Industry (TAPPI,
Lignin peroxidase (LiP) and manganese peroxidase (MnP) activities were measured
according to the procedure of Castillo et al. (1994)
using the substrate know as 0.167 mM 3-methyl-2-benzothiazolinone hydrazone
(MBTH) upon mixing with 2.37 mM 3-(dimethylamino) benzoic acid (DMAB) to produced
a purple colour solution. Substrates were prepared using succinic-lactic acid
buffer at pH 4.5. 3 mM of MnSO4 were used in order to determine the
dependency of manganese and peroxidase in the culture. Reaction were carried
out at 37°C and monitored by spectrophotometer at 590 nm for a min.
The lignin content of OPEFB after alkaline pretreatment using 2% NaOH is
shown in Table 1. After soaking OPEFB in 2% NaOH solution
at temperature 90°C, the kappa number measured was 61.32 and lignin content
(Klason lignin) was 7.97%. As time increase to 3 h the kappa number decrease
to 45.59 and the lignin content was 5.93%. For untreated OPEFB, kappa number
could not be obtained due to high content of lignin.
For microbial pretreatment, two type of cultivations mode was done, solid
state and liquid fermentation. The lignin content of OPEFB after 3 days pretreatment
using Phanerochaete chrysosporium ATCC 32629 in solid fermentation medium
was 8.30% (Table 1). As the time of fermentation increased
to 7 days the lignin content decreased to 5.89%. Comparing with liquid fermentation
medium, the lignin content obtained after 7 days fermentation was 6.22%.
The higher delignification after 7 days fermentation was due to higher production
of total peroxidase, lignin peroxidase and manganese peroxidase by the fungus
(Fig. 1). The activities of total peroxidase, lignin peroxidase
and manganese peroxidase at day 3 were 3.212, 2.847 and 0.365 U mL-1,
respectively, while the activities of total peroxidise, lignin peroxidase and
manganese peroxidase at day 7 were 3.521, 2.616 and 0.905 U mL-1,
respectively. In liquid fermentation, the fungus produced a much lower activities
of total peroxidase, lignin peroxidase and manganese peroxidase (Fig.
2). The activities of total peroxidase, lignin peroxidase and manganese
peroxidase at day 3 were 0.812, 0.501 and 0.311 U mL-1, respectively
while the activities of total peroxidase, lignin peroxidase and manganese peroxidase
at day 7 were 1.115, 0.495 and 0.620 U mL-1, respectively. Result
of this study showed that lignin peroxidase production in solid state fermentation
(Fig. 1) is higher compared to liquid fermentation (Fig.
||Kappa number, klason lignin and percentage of lignin removal
for chemical and microbial pretreatments
|aKappa number estimation was based on titration
method (TAPPI, 1999)
bKlason lignin is a measurement of percentage residual lignin
by equating the Kappa number with factor 0.13 (TAPPI,
1999). Hence, percentage of delignification can be quantified. cAssuming
lignin content in OPEFB at 20.4% (Khalid et al.,
||Peroxidase activity in liquid fermentation
Two methods of pretreatment were attempted: Chemical pretreatment using simple
heating in 2% (w/v) NaOH and microbial pretreatment using Phanerochaete chrysosporium
ATCC 32629. For microbial pretreatment, two techniques were studied, solid state
and liquid fermentation. Klason lignin and Kappa number (TAPPI,
1999) were used as an extent measurement of delignification processes. These
quantification techniques were usually used in pulp and paper industries to
evaluate the extent of delignification and residual lignin remaining in the
cellulosic fractions prior to paper-making processes.
Compared with acid pretreatment, alkaline pretreatment with NaOH removed more
lignin fraction from the biomass because of the solubilisation of lignin in
alkaline solution (Chen et al., 2009). Delignification
still can be achieved by using microbial method. Excreting of unique extra-cellular
peroxidase (LiP and MnP) from Phanerochaete chrysosporium can decay lignin
efficiently (Tien and Kirk, 1988). The growth and metabolism
of lignin degrading microbe Phanerochaete chrysosporium shows a great
effect on microbial pretreatment (Shi et al., 2009b).
Nutrients supplementation and cultivation conditions affect the production of
ligninolytic enzymes during cultivation of the microbes are depend on nutrients
supplementation and cultivations conditions. So, the production of the enzymes
still can be increase by optimization study. According to Brown
et al. (1990) the balanced addition of trace metal, such as Mg2+,
Ca2+ and Mn2+ was important for ligninase production.
From this study, it showed that higher delignification occurs in solid fermentation
as compared in liquid fermentation. Solid state fermentation shown higher efficiency
in delignification process due to environmental conditions being more similar
to those in nature (Shi et al., 2009a).
Although, microbial pretreatment method using Phanerochaete chrysosporium
ATCC 32629 could delignify OPEFB at the same level as the alkaline pretreatment
method, alkaline pretreatment was preferred method, as only 3 h were required
to achieve preferable delignification level. Reducing the lignin content is
important to expose the crystalline structure of cellulose. However, less time
consumed for chemical pretreatment means it is quite harsh for the substrate.
According to Hendriks and Zeeman (2009) the result of
the pretreatments is dependent on the biomass composition and operating conditions.
As a result, microbial pretreatment showed significant of lignin removal, but
timely as compared to chemical pretreatment.
Delignification can be achieved either by chemical pretreatment using 2% NaOH or via microbial pretreatment using Phanerochaete chrysosporium ATCC 32629. However, in microbial pretreatment, lignin removal is dependent on the microbial ability either to consume lignin or to produce biological products such as enzymes, to remove lignin. Microbial pretreatment can be used as a gentle process yet, longer time is required (7 days). Chemical pretreatment using 2% (w/v) NaOH can be used as an alternative process of delignification. Furthermore, similar percentage of lignin removal was achieved within less than 3 h.
The authors would like to thank the Ministry of Science Technology and Innovation of Malaysia for financially support throughout this research project.