Empty Fruit Bunches (EFB) fibre, one of the lignocellulosic material consist
primary of cellulose and hemicellulose, is an alternative to the traditional
feedstock for lactic acid production. Theoretically, utilization of the lignocellulosic
material as a feedstock in the organic acid production can decrease production
cost, since it is inexpensive and widely available renewable carbon source that
has no competing food value. The choice of the feedstock depends on its price,
availability and on the respective product recovery and purification costs (Datta
et al., 1995). According to Tejayadi and Cheryan
(1995), the cost for raw material possessed 68% of the total cost for lactic
acid production. Previous study on the simulation of lactic acid fermentation
process by Akerberg and Zacchi (2000) reported that
the operational cost including raw material, neutralizing agent, hydrolyzing
enzyme and membrane for electrodialysis contributed to approximately 80% of
the total cost for lactic acid production. Since, raw material cost cannot be
reduced by scaling up process, EFB has been considered as attractive substrate
for lactic acid production.
The main pathway to derive fermentable sugar from EFB is through enzymatic
hydrolysis by cellulolytic and hemicellulolytic enzyme. A mechanical and chemical
pre-treatment of the lignocellulose is required to reduce particle size, to
modify and or to remove lignin and with that to enhance the accessibility of
the polysaccharide for enzymatic hydrolysis (Akerberg and
Zacchi, 2000; Moiser et al., 2005). Various
pre-treatment methods of lignocellulose have been studied for conversion of
lignocellulose to organic acid. One of the promising technologies is microwave
alkaline (MW-A) pre-treatment. Through this pre-treatment technique, enzymatic
digestibility of the fibre is increased due to the disruption of the recalcitrant
structure of the fibre (Hu and Wen, 2008). Therefore,
the cellulose molecule is more exposed to the enzymatic attack and thus encouraged
the conversion to soluble sugar that can be used in the fermentation of lactic
Lactic acid has gained prominence in research and industry because of its potential
for biodegradable and biocompatible lactic acid polymers. Polylactate polymers
such as Poly Lactic Acid (PLA) could be an environmental friendly alternative
to plastics derived from petrochemical materials. Due to the unique properties
of PLA, lactic acid has the potential to be a substitute for biodegradable plastics
and becomes a very large volume commodity chemical intermediate (Huang
et al., 2005). On the other hand, antitumor and antimicrobial effects
of PLA have also been reported.
Lactic acid occurs naturally in two isomers, L-lactic acid and D-lactic acid. However, elevated levels of the D-isomer are harmful to human. Thus, L-lactic acid is the preferred isomer in food and pharmaceutical industries. The most commonly used lactic acid is synthesised from chemical route which involved the hydrolysis of lactonitrile. Unfortunately, lactic acid produced is in the racemic form whose resolution is difficult. Thus, the direct microbial synthesis of the pure isomer is preferable.
One of the potential microbe for this purpose is filamentous fungi Rhizopus
oryzae because of its outstanding ability to directly produce almost optically
pure L(+)-lactic acid with low nutrient requirement and high yield (Yin
et al.,1997; Rosenberg and Kristofikova, 1995).
Rhizopus oryzae can be grown in submerged cultures in several different
morphological forms such as; pellet, suspended mycelium or clump. Morphological
differences may have a significant influence on the formation of metabolic product
(Couri et al., 2003). If the fungal cells grow
as mycelia or large pellets or clumps, large scale production of lactic acid
could be difficult because the growing mycelia will result in highly viscous
broth and thus limit the oxygen transfer inside the cell (Zhou
et al., 1999).
Thus, an attempt was made to investigate the productivity and yield of lactic acid from MW-A pre-treated EFB in SSF using two different morphologies of Rhizopus sp. Performance of clumps and pellets Rhizopus on lactic acid was compared. Since, the size of the pellet produced is not uniform for all of the samples, thus in the present study the Aspect Ratio (AR) is introduced to identify the perfect pellet produced during cultivation.
MATERIALS AND METHODS
Substrate preparation: EFB obtained from Seri Ulu Langats palm oil milling, Dengkil, Selangor, Malaysia was dried and grinded into 1 mm particle size using IKAE grinder (German). The composition of hemicellulose and lignin was determined by triplicates using Acid Detergent Fibre (ADF), Acid Detergent Lignin (ADL) and Neutral Detergent Fibre (NDF). Meanwhile, the composition of cellulose was calculated from the deduction of ADF with ADL value.
Microwave pre-treatment: Pre-treatment of EFB particles was conducted
in a microwave oven model (NN-5626F) with the following specification: rated
power output (240v-50 Hz) operation. In the pre-treatment process, 2.5 M of
sodium hydroxide solution was used and the process was performed in a round
bottom glass vessel set up completef with a stirring (Ani
and Iqbal, 2008). During the pre-treatment, the agitator was set at 150
rpm. Prior to microwave treatment, the EFB were pre-soaked with sodium hydroxide
solution at room temperature for 2 h. The pre-soaked slurry was then transferred
to the round bottom flask reactor vessel and treated in the microwave oven.
The power setting was set to medium. The EFB were exposed to irradiation for
1 h reaction time. When the microwave irradiation pre-treatment was completed,
the reaction vessel was removed from the microwave oven and cooled to the room
temperature. The slurry in the reaction vessel was then filtered through a Whatman
filter paper No. 1. The filter cake was washed with deionized water for a few
times to neutralize the pH to 7.0, dried and stored for the enzymatic hydrolysis.
Pellet preparation: Rhizopus oryzae NRRL 395, a (L)-lactic acid producing strain was a gift from USDA (Northern Regional Research Centre, United State Department of Agriculture, Peoria, Illinois). The strain was maintained on Potato Dextrose Agar (PDA) for fermentation study, agar slant containing fungus was washed by sterile water to obtain spore suspension. Spore suspension of 1x102 spores mL-1 was used for production of the pellet Rhizopus. The pre-culture composition of the pellet formation medium was as follows (g L-1): glucose, 50; KH2PO4, 0.2, ZnSO4.7H2O, 0.04; MgSO4.7H2, O 0.25 and (NH4)2SO4, 2.0. The pH of the medium was adjusted to 6.0 before steam sterilization. The cultivation temperature was set to 37°C with agitation of 170 rpm. This cultivation process is continued for 3 days so as to allow the formation of pellet/clump Rhizopus.
Simultaneous saccharification and fermentation of lactic acid from MW-A pre-treated EFB: For production of lactic acid, 15 g L-1 of MW-A pre-treated fibre was used in 250 mL Erlenmeyer flasks containing 50 mL of fresh medium. The medium (g L-1) used in SSF process consisted of KH2PO4, 0.2, ZnSO4.7H2O, 0.04; MgSO4.7H2O, 0.25 and (NH4)2SO4, 1.35. The pH of the medium was adjusted to pH 6.0. All flasks were incubated in the orbital shaker with agitation of 170 rpm. The culture temperature was maintained at 37°C throughout the experiments. The reduction in pH of the medium was controlled by addition of 5% CaCO3. Culture was carried out for 4 days and the sampling was done every 24 h. Each experiment was repeated in triplicate and presented as an average.
Analysis: At specific intervals of time, samples were taken and then
heated in boiling water to denature the enzyme. The heated samples were centrifuged
at 5000 rpm for 5 min and supernatants were filtered through 0.45 μm syringe
filter. Finally, the glucose and lactate concentration were measured by glucose
analyzer (YSL instrument). Lactate yield was defined as amount of lactate produced
divided by total amount of pre-treated EFB used and productivity as lactate
concentration divided by fermentation time.
Aspect ratio determination: The morphology of the cultures was determined
by examining submerged cultures dispersed on Petri dishes. A microphotographer
(Olympus) was used to observe the pellet morphology and measured the size of
the pellets. Prior to processing of images, care was taken to assure that all
pellets were detected as single entities. The maximal diameter (Dmax),
minimal diameter (Dmin) and average diameter of the pellets was determined.
The values obtained were used to calculate the Aspect Ratio (AR) of the pellet
using the following equation.
The dry biomass was determined by neutralizing the excess CaCO3
using 6 N HCL, washed using distilled water and dried at 80°C for 24 h before
RESULTS AND DISCUSSION
EFB composition after MW-A pre-treatment: The composition of the raw and MW-A pre-treated EFB were shown in Table 1. The results revealed that the EFB fibre which was subjected to MW-A pre-treatment showed a reduction in the composition of the hemicellulose and lignin; however the percentage of cellulose obtained was increased to 74.3%. The percentage of the hemicellulose was reduced from 35% in raw EFB to 16.8%, while, the lignin decreased from 16.4 to 7.6% when subjected to MW-A pre-treatment. The increment in cellulose percentage obtained is probably because of the microwave heating technique which contributed to the effective removal of lignin and hemicellulose.
In microwave, heating results from the interaction of the electromagnetic wave
with the irradiated medium. Principle of microwave heating is based on molecular
friction or dielectric loss. The material molecules are stimulated and rotated
millions of times a second in response to electromagnetic field and these rotations
quickly generate heat in the material in a manner similar to friction.
||Compositions of raw and EFB pre-treated with MW-A
Such rapid intense heating caused the rupture of the lignin and hemicellulose
structure. It is a mass heating where heat transfer occurs from the treated
medium to the outside.
During microwave pre-treatment, the heat produced selectively heats the more polar (lossy) part and creates hot spots within the inhomogeneous EFB. In addition, the presence of agitator in microwave cavity distributed a uniform heating within the reaction vessel. It is hypothesized that this unique heating feature results in an explosion effect among the particles and improves the disruption of the recalcitrant structures of lignocellulose. Hydrogen bonds between hemicellulose and cellulose were ruptured during the treatment and thus, reduced the stability form between lignin-hemicellulose-cellulose matrixes.
Consequently, more cellulose was exposed after microwave pre-treatment process
as observed in Table 1. Additionally, combination of NaOH
with microwave pre-treatment reduced the crystallinity of the cellulose in the
EFB. According to the Thostenson and Chou (1999), the
effectiveness of the microwave pre-treatment is dependent on the material behavior.
In fact, the crystallinity of the fibre affects the dielectric properties in
the microwave pre-treatment. Degrees of crystallinity above 45% are essentially
transparent to microwaves due to the restriction of dipoles. Addition of NaOH
as a swelling agent during microwave treatment helps in the formation of more
disorganized amorphous cellulose structure. As been reported by Jacobsen
and Wyman (2000), approximately 50-90% of the total cellulose is the crystalline
structure where the remainder is the disorganized amorphous cellulose. However,
the amorphous cellulose is more rapidly hydrolyzed in enzymatic reaction than
the crystalline regions. Here, NaOH solution acts an intracrystalline swelling
agent that is capable to penetrate and swell both the accessible amorphous and
crystalline region (Shujun et al., 2007). At
the same time, destruction of cellulose crystalline structure occurred and the
highly ordered fibrils in cellulose were distorted. As a consequence, the microfibrils
were separated from the initial connected structure and fully exposed, thus
increasing the external surface and the porosity of the cellulose.
Aspect ratio and morphology: In cultivation of filamentous Rhizopus,
the spores tend to aggregate and grow as pellets which have a variety of compactness.
Pellets are spherical or ellipsoidal masses of hyphae with variable internal
structure, ranging from loosely packed hyphae, forming fluffy pellets, to tightly
packed, compact, dense pellets.
||Rhizopus pellet with AR = 1.0
||Rhizopus pellet with AR = 1.5
In the present study, AR was used to characterize
pellet morphology since the size of the pellet produced during cultivation is
not uniform in size. AR of 1 indicated a prefect pellet whereas a value closer to 1 indicated rounder
pellets. The perfect Rhizopus pellet with AR = 1 obtained is illustrated
in Fig. 1. On the other hand, AR of 1.5 mostly showed the
ellipsoidal pellet as shown in Fig. 2. The results indicated
that the ellipsoidal pellet is bigger in size (>3 mm) compared to the spherical
In term of quantities, the pellets with AR more than 1.5 were produced in fewer quantities. Meanwhile, AR failed to classify the clump morphology because the clump morphology tends to aggregate with other segment thus forming a long structure (Fig. 3).
The morphology of a filamentous fungus developed in any fermentation system could be considered as a final result of competing influence, equilibrium between forces of cohesion and disintegration. Shear forces may be unambiguously assigned the role as disintegrating factors. At pH value above 5.5, cell walls of most microorganisms are negatively charge, tending to cause separation or cell aggregation by electrostatic repulsion.
||Clump morphology of Rhizopus oryzae NRRL 395
This may be suppressed by an increase in ionic strength or bridging cells with
Ca2+ ions. Addition of polycations usually induces aggregation whereas
polyanion suppress it (Domingues et al., 2000).
The surface thermodynamic balance fungal cell and liquid medium was found to
be responsible for pellet formation since Gibbs free energy of pellet formation
of the initial culture media -73 to -81 ergs cm-2 were increased
to -13 to -46 ergs cm-2 at 48 h. The factors inducing pellet formation,
simultaneously increased the cell wall hydrophobicity (Papagianni,
L-lactic acid concentration and productivity: In the present study,
SSF of the lactic acid from MW-A pre-treated EFB using Rhizopus oryzae
was studied. The results indicated that Rhizopus oryzae NRRL 395 has
an ability to perform a single stage SSF process for lactic acid production
using cellulosic material. SSF with clump or pellet Rhizopus selectively
produced L-lactic acid with a high concentration. The production of lactic acid
in SSF took place simultaneously with the hydrolyzed cellulose to soluble sugar.
Rhizopus sp.has a high enzymatic and metabolic capability to perform
an SSF process and also utilize cellulose as a carbon source for lactic acid
production (Karimi et al., 2006).
The MW-A pre-treatment of the EFB made cellulose molecule in the fibre easily
accessible to metabolite activities of the fungus. From the experiments performed
it was observed that after 24 h of fermentation, the cultivation medium became
clear when most of the EFB fibre was coagulated with the pellets. Along with
this mechanism, the diameter and density of the pellet increased with the time
and this condition represent the increment in the biomass produced during fermentation.
As been reported by Huang et al. (2005) fungal
cell growth appeared to be very fast, resulting to a sharp increase in fungal
biomass production. Associated with the fungal biomass produced, the lactate
yield was excreted as a metabolite product and the results are illustrated in
Under similar cultivation conditions, the pellet Rhizopus demonstrated
a higher lactate yield than clump Rhizopus. Lactate production in the
pellet cultivation is almost 30% higher than in clump system.
||Productivity of L-lactic acid in SSF using clump and different
AR Rhizopus pellet
||Lactate yield in the SSF of MW-A treated EFB using pellet
(AR = 1) and clump rhizopus
In the cultivation of medium containing approximately 15 g L-1
MW-A pre-treated EFB, pellet Rhizopus with AR = 1 produced lactate at
0.77 g g-1 EFB within 96 h, while with clump Rhizopus, yield
of the lactate is approximately 0.57 g g-1 EFB. Pellet Rhizopus
with AR = 1 also gave the highest productivity of lactic acid as compared to
the clump and pellet with AR>1.5 (Table 2, Fig.
4). Productivity of the lactic acid obtained using pellet with AR = 1 was
0.12 g Lh-1 Meanwhile, clump and pellet with AR 1.5 cultivation gave
productivity of 0.089 and 0.099 g Lh-1, respectively.
Size of the pellet is an important factor in metabolite activity of the fungus.
As reported by EL-Enshasy et al. (1999) smaller
pellets were more efficient with respect to the production of exocellular glucose
oxidase by Aspergillus niger. Generally, the pellets with AR = 1 are
smaller in size compared to the pellets with AR = 1.5. The decreasing pellet
size also correlated with an increased mycelia density, indicating an improvement
of the transport of nutrient to the inner part of the pellet. According to Couri
et al. (2003), higher diameter pellets tend to suffer autolysis of
the cells in their inner diameter, particularly if their cores are also large
making it difficult for the nutrients to be transport to the core.
It is known that mineral or nutrient deficiency in fungi growth cause a decrease
in the activity of several enzymes in the fungi metabolite. Meanwhile, for the
clump morphology, the high hyphae entanglement had probably produced a compact
core where the inner part was lysed due to a deficient nutrient transportation.
The high annular area of the pellet could not compensate this effect and the
enzyme activity was low (Hermersdorfer et al., 1987).
Thus, the metabolite activity is reduced, thus reducing the productivity of
the lactic acid in SSF reaction.
Microwave-alkali (MW-A) pre-treated EFB has been used as a carbon source in the SSF of the lactic acid using Rhizopus oryzae NRRL 395. Cellulose molecules in the EFB were converted by Rhizopus metabolite into lactic acid. The lactic acid production from SSF of MW-A pre-treated EFB is very much influenced by the different morphologies of Rhizopus used. Pellet with as AR = 1 indicated a perfect pellet while aspect ratio of 1.5 represented an ellipsoidal pellet. Lactic acid yield and productivity was the highest when SSF was cultivated with pellet Rhizopus than clump Rhizopus. The maximum lactate yield was 0.77 g g-1 EFB used after 96 h cultivation. Highest productivity of the lactic acid (0.12 g L-1) was obtained from SSF of MW-A pre-treated EFB using pellet (AR = 1).
The present research was made possible through IRPA grant sponsored by the Ministry of Science, Technology and Innovation of Malaysia (MOSTI), UiTM scholarship and also facilities in Faculty of Chemical Engineering and Natural Resources Engineering, Universiti Teknologi Malaysia (UTM).