Citric acid is one of the largest fermentation products which is widely used
in the food processing, beverage, cosmetic, pharmaceutical, chemical, textile
and electroplating industry (Tran et al., 1998)
and bioremediation of heavy metal contaminated soil (Kim
and Barrington, 2003). The demand of citric acid is increasing from 4-5%
every year (Pandey et al., 2001; Vandenberghe
et al., 2000) that can only be met economically by using less expensive
and renewable agro industrial residues as raw materials through solid state
bioconversion with appropriate microorganism (Tran et
The oil palm Empty Fruit Bunches (EFB) as a lignocellulosic residue is produced
from oil palm industries, which are abundantly available in Malaysia. The annual
production of the EFB 12.4 million tones (Tanaka et al.,
2006) which shows a great challenge to the solid waste management for its
safe, scientific and environment friendly disposal (Bari
et al., 2009; Suhaimi and Ong, 2001). Lignocellulosic
residues are very well adapted to solid-state cultures due to their cellulosic
and starchy nature (Soccol and Vandenberghe, 2003).
Therefore, oil palm EFB was considered to be a potential alternative renewable
raw material for the citric acid production by solid state bioconversion with
The investigation on particle size of different lignocellulosic residues has
been carried out by different researchers for citric acid production using different
strains of Aspergillus niger through solid state fermentation. The lignocellulosic
residues such as cassava bagasse (Prado et al., 2005a,
b), sugarcane bagasse (Kumar et
al., 2003a), carob pod (Roukas, 1999), coffee
husk. Shankaranand and Lonsane (1994) have been examined
by grinding in different particle size to evaluate the effect on production
of citric acid by solid state bioconversion.
From the above discussions, it is clear that the particle size of substrate is an important parameter for the citric acid production from lignocellulosic substances. Therefore, the aim of this study is to evaluate the effect of particle size of oil palm empty fruit bunches towards the enhancement of production of citric acid through solid state bioconversion with wild strain of Aspergillus niger.
MATERIALS AND METHODS
Microorganism and inoculum: Wild strain of Aspergillus niger
IBO-103MNB (IMI396649) was used for this study by selecting through screening
test among 26 locally isolated strains from two citrus fruits viz., lemon and
orange. The strains were maintained at 4°C by sub-culturing every month
for further use. The inoculum was prepared by washing spores grown on PDA plate
after 4 days of culture growth with 25 mL sterilized distilled water and collected
in 100 mL Erlenmeyer flask by filtering with Whatman No. 1 filter paper. The
spores were counted by haemocytometer to maintain the concentration of inoculum
at 3x108 spores mL-1.
Collection and pretreatment of empty fruit bunches: Sample of Empty
Fruit Bunches (EFB) was collected from Seri Ulu Langat palm oil mill in Dengkil,
Selangor, Malaysia and preserved in cold room at 4°C to avoid the unwanted
bio-degradation by any microorganisms. The EFB samples were washed vigorously
with tap water to ensure the removal of all unwanted impurities followed by
drying at 105°C for 24 h. EFB fiber was split out manually and then ground
by cutting mill to obtain the particle size of 0.5 mm down graded, 0.5 to 1
mm and 1 to 3 mm. Ground EFB was dried at 60°C for 48 h to get constant
dry weight for experimental study (Kim and Barrington, 2003;
Kumar et al., 2003b). The effect of different
particle size was evaluated on the basis of production of citric acid.
Fermentation media: Twenty gram of media containing 5 g (25% w/w) of
major substrate-treated and non-treated EFB (particle size = 0.5 mm) with 1
g (5% w/w) of sucrose and 1 mL (5% v/w) of mineral solution obtained with modification
from Erikson et al. (1980)
containing NH4H2PO4 (2 g L-1), KH2PO4
(0.6 g L-1), K2HPO4 (0.4 g L-1),
MgSO4.7H2O (0.5 g L-1), CaCl2.2H2O
(74 mg L-1), Ferric acid citrate (12 mg L-1), ZnSO4.7H2O
(6.6 mg L-1), MnSO4 (5 mg L-1), CuSO4.5H2O
(1 mg L-1).
Experiment for solid state bioconversion: The experiment for solid state bioconversion was carried out in 250 mL Erlenmeyer flask with initial moisture content of 70% by weight adjusted with mineral solution, distilled water and inoculum. The sample was inoculated with 5% spore suspension of 3x108 spores mL-1 (inoculum) after autoclaving at 121°C for 15 min. The initial pH of the substrate was recorded 5.5 but was not adjusted during the experimental runs. Bioconversion was carried out for 2, 4, 6, 8 and 10 days by incubating at 32°C. All SSB experiments were conducted in triplicates.
Extraction and analysis of citric acid: Citric acid was extracted from
fermented substrate by adding 50 mL distilled water and shaking for 1 h at 150
rpm at room temperature (28±1°C) (Tran et
al., 1998). The supernatant was collected by filtering with Whatman
No. 1 filter paper and immediately analyzed to determine the content of citric
acid and remaining sugar. The concentration of citric acid in extraction was
determined by using spectrophotometer at 420 nm using pyridine-acetic anhydride
method as suggested by Marrier and Boulet (1958).
RESULTS AND DISCUSSION
Effect of particle size of EFB on citric acid production: The experimental results of five different particle size of EFB are presented in Fig. 1 to evaluate their effect in terms of maximum production of citric acid using lignocellulosic material (EFB) as new substrate by solid state bioconversion. The effect of particle size on citric acid production was clearly observed during the bioconversion period. Figure 1, it is observed that EFB with particle size of 0.5 mm gave higher production of citric acid during the bioconversion period compared to EFB of 0.25, 1, 2 and 3 mm down graded particle size. The maximum production of citric acid from EFB with particle size of 0.5 mm was 131.3 g kg-1 of dry EFB with the production rate of 14.6 g kg-1-day obtained after 8 days of bioconversion. However, production of citric acid of 124.9 g kg-1 of dry EFB obtained after 6 days of bioconversion with the production rate of 20.8 g kg-1-day from the same article size of EFB. The lowest citric acid production was obtained from EFB of 3 mm particle size. Citric acid production from EFB with particle size of 0.25 mm was very close to 1 mm particle size until 8 days of bioconversion.
Higher production of citric acid from smaller particle size was presumably
due to easily availability of substrate to the microbe and the substrate with
0.5 mm particle size exhibited optimum porosity that can provide better heat
and mass transfer (Kumar et al., 2003a).
||Effect of particle size of EFB on production of citric acid
|| Consumption of sucrose during the bioconversion
On the other hand, larger particle sizes of EFB reduced substrate availability
to the microbe though it increased the mass and heat transfer with higher inter
particular spaces. Similarly, lowest particle size of 0.25 mm was more compacted
compare to other sized that caused inadequate heat and mass transfer.
The obtained maximum production of citric acid from particle size of 0.5 mm
was higher than the citric acid production of 121 g kg-1 from sugarcane
bagasse with particle size of 1.2-1.6 mm obtained by Kumar
et al. (2003a) and lower than the citric acid production of 176±4
g kg-1 from carob pod with particle size of 0.5 mm obtained by Roukas
(1999). Therefore, the highest productions of citric acid in laboratory
scale from EFB with particle size of 0.5 mm are considerably comparable with
the production of citric acid from other lignocellulosic substrates reported
by different researchers.
Consumption of sucrose by Aspergillus niger IBO-103 MNB grown on different particle sizes of EFB during the bioconversion is presented in Table 1. The result shows that the highest consumption of 171.7 g kg-1 of dry EFB was taken place during the bioconversion of EFB with particle size of 0.5 mm down grade. The result also depicted that the consumption of sucrose was gradually less for higher and for lower particle size compared to 0.5 mm. These phenomena could be explained by the fungal growth during the bioconversion. The maximum consumption is meaning that maximum utilization of sucrose to citric acid through bioconversion.
The comparison of fungal growth in terms of protein content shows that the highest growth was accomplished on 0.5 mm particle size of EFB (Fig. 2). On the other hand, lowest growth was found on particle size of 3 mm. The highest protein content was found of 38 g kg-1 of dry EFB after 6 days of bioconversion of 0.5 mm particle size of EFB. This result indicated that the particle size of 0.5 mm is the best for the growth among the particle size studied.
The evolution of pH for all ranges of particle sizes followed same trend and
differences of pH values were not found among the experiments of different particle
sizes. Figure 3 shows that the pH values sharply dropped to
around 2.65 after 2 days of bioconversion and almost remain constant at around
2.3 from 4 days to the end of bioconversion.
|| Growth of fungi on EFB with different particle sizes
||Evolution of pH during bioconversion of EFB of different particle
However, the lowest pH was observed for particle size of 0.5 mm though the
difference was very less.
Effect of particle size distribution: Inter particular spaces or porosity of substrate that depends on the particle size distribution is one of the parameters that govern the mass and heat transfer during the bioconversion of substrate. The 0.5 mm down graded EFB particle was evaluated with three particle size distributions, such as well graded, uniformly graded and gap graded. The particle size distribution is presented in Fig. 4 where particle sizes are plotted along the x-axis in logarithmic scale against the percent finer along the y-axis in normal scale.
Well-graded sample is composed of different percentages of all particle sizes
that forms an S-shaped curve. In case of uniformly-graded sample, all the particle
sizes participate in same percentage while gap-graded sample dose not represents
all sizes of particles. In this study, representative samples of particle sizes
of 0.212 and 0.106 mm were absent from the total sample.
|| Particle size distribution in different samples
||Production of citric acid with different graded particle sizes
In this case, 95% of total sample was finer than 0.425 mm and then 35% was
finer than 0.075 mm which is obviously finer than 0.212 and 0.106 mm. Four samples
of EFB particle were tested as well-graded-1, well-graded-2, uniformly-graded
and gap-graded. Well-graded-1 particle size distribution was obtained naturally
in ground EFB sample. Well-graded-2 was prepared by adding at certain percentage
of different finer particles to evaluate the difference. Similarly, uniformly-graded
and gap-graded samples were prepared with certain composition.
Figure 5 shows that the highest production for all the particle size distribution was obtained after 8 days of bioconversion. The well-graded-1 and well-graded-2 gave the higher production of citric acid compared to well-graded and gap-graded samples. The productions of citric acid between the well-graded samples were almost same through well-graded-2 sample showed slightly better result with the production of 134.3 g kg-1-EFB while, 133.2 g kg-1-EFB was obtained from well-graded-1 sample. This slight variation might be due the variation of substrate accessibility to the microbes as well as heat and mass transfer.
Figure 4 shows that well-graded-2 sample was prepared with higher percentage of smaller particles that provided the higher substrate accessibility. This phenomenon might enhance the production. However, higher percentage of smaller particles reduced the porosity of the substrate that hampered the heat and mass transfer as well as production of citric acid. On the other hand, well-graded-1 sample was composed of comparatively lower percentage of smaller particle that might provide slightly different situation than well-graded-2 sample for bioconversion.
The productions of citric acid of well-graded samples were higher than uniformly-graded and gap-graded samples might be due to the same reasons i.e., well-graded samples were able to provide optimum substrate accessibility as well as heat and mass transfer conditions. The heat and mass transfer ability of uniformly-graded and gap-graded samples might be better than well-graded samples but substrate accessibility was definitely less. Similar reasons were also applicable to justify the difference of production between uniformly-graded and gap-graded samples.
The result obtained in this study indicated that the production of citric acid increased with the decrease of particle size of EFB up to a certain level. The maximum citric acid production was 133-134 g kg-1-EFB obtained from well-graded of 0.5 mm down graded particle size of EFB after 8 days of bioconversion with the production rate of 16.62-16.75 g kg-1-day. However, the highest production rate of citric acid was 21.6 g kg-1-day obtained by producing 129.6 g kg-1-EFB after 6 days of bioconversion from the same particle size of EFB. Therefore, the well-graded of 0.5 mm down graded particle size of EFB might be the effective for further studies.
The authors are grateful to the Department of Biotechnology Engineering, International Islamic University Malaysia for their support and to the Ministry of Science, Technology and Innovation (MOSTI), Malaysia, for financing the research project (No. 02-01-08-SF0050) under eScience research grant.