SCHOOL OF BIOPROCESS ENGINEERING

UNIVERSITY MALAYSIA PERLIS

ERT 317: BIOCHEMICAL ENGINEERING
EXPERIMENT 4: EFFECT OF CELL IMMOBILIZATION ON DYE REMOVAL BY
BIOABSORBENT

GROUP NO.

: A10

GROUP MEMBERS

: CHANG LEE WEI (141141152)

ROY NICHOLAS A/L XAVIER SELVARAJ (141141252)
ZAHIRAH BINTI MD ISA (141141221)
NURUL NADIAH BINTI SHAHRIR (141141201)

DATE OF EXPERIMENT: 4TH NOVEMBER 2016

DATE OF SUBMISSION: 11TH NOVEMBER 2016
LECTURER:

: DR SALEHA SHAMSUDIN & MDM HAFIZA SHUKOR

PLV

: MR AHMAD RADI WAN YAAKUB

1.0 OBJECTIVES:
1.1 To immobilize fungal biomass on activated carbon for bioabsorbant.
1.2 To analyze the effect of cell immobilization for methylene blue dye removal by bioabsorbent.
2.0 THEORY:
Various dyes have been used in textile, dyeing, paper pulp, plastic, leather, cosmetics and
food industries. The effluents produced from these industries possess certain hazards and
environmental problems. Most of these dyes are synthetic and exhibit complex aromatic
structure. Thus, they are stable and difcult to be biodegraded. A common example of dyes is
methylene blue. Methylene blue is a heterocyclic aromatic chemical compound that is very
stable to light, oxidizing agents and resistance to aerobic digestion. So, these effluents have
to be treated before discharge not only for their high chemical and biological oxygen demand
but also for suspended solids of toxic properties to aquatic life.
Several techniques have been developed to treat dye efuents such as microbial
degradation, chemical oxidation, ion exchange, membrane separation, bioaccumulation,
electrochemical treatment, adsorption and reverse osmosis. Adsorption is generally preferred
among these techniques due to its high efciency, easy handling, low energy input and
availability of different adsorbents. The most commonly used adsorbent for dye removal was
activated carbon. Activated carbon is carbon that has been treated with oxygen to open up
millions of tiny pores between the carbon atoms. The highly porous charcoals have huge
surface areas that give it countless bonding sites. As a result, activated carbons are widely
used to adsorb odorous or coloured substances from gases or liquids.
The adsorption process is one of the efficient methods to remove dyes from effluent and
has an advantage over the other methods due to the excellent adsorption efficiency of
activated carbon (powdered or granular) for organic compounds even from dilute solutions,
but commercially available activated carbons are very expensive. The high adsorptive
capacities of activated carbons are related to properties such as surface area, porosity, and
surface functional groups. These unique characteristics are dependanton the type of raw
material employed and method of activation. Basically, there are two different processes for
the preparation of activated carbon: physical and chemical activation (Ahmadpour & Do,
1996).

Activated carbon has also some disadvantages. Both chemical and thermal
regeneration of used carbon is expensive,impractical on a large scale and produces additional
effluent and results in considerable loss of the adsorbent. It is also documented that
adsorption with activated carbon and other technology like chemical precipitation was
ineffective when the pollutant concentration range from 1 to 100 mg/L. On the other hand,
ion exchange and membranes separation were effective at the range of 1100 mg/L, but it
was extremely expensive in treatment cost. Furthermore,some of these methods would
produce huge quantity of toxic chemical sludge that required the secondary treatment (Li,
Liu, Li, & Deng, 2008).
Recently, several researchers have shown that biosorption can be regarded as a valid
alternative to chemical-physical method and to microbial and/ or enzymatic biodegradation. Such
researches have pointed out the capacity of various microbial biomass (bacteria, yeast, fungi, and
algae) and waste materials from industry and agriculture to absorbor accumulate dyes (Crini,
2006; Ferrero, 2007). Among the various types of biomass, the fungal biomass has proved to be
particularly suitable. Even if the mechanisms regulating biosorption have not yet been fully
explained, it seems to take place at the cell wall level. The main attractions of biosorptions are its
high selectivity and efficiency, good removal from large volumes and the potential cost
effectiveness. Moreover, both living and dead biomass can be used to remove hazardous
organics; dead cells are obviously preferable for wastewater treatment since they are not affected
by toxic wastes and chemicals and do not pollute the environment by releasing toxins and/or
propagules ( Aksu & Donmez 2005). However, dead and dried biomass can be stored for long
periods at room temperature with little risk of putrefaction. This makes it easier to use and
transport. Furthermore, these biosorbents are usually used in free forms, which would bring
about the difficulty of separation between solid and liquid after adsorption. The traditional
technologies of separation are adopted such as centrifugation and membranes separation. But it is
generally difficult in continuous process for wastewater treatment. Even though separation would
be successful, additional steps must result in the increase of cost.
Recently, the immobilization of active compounds onto microbial biomass is an
interesting technique to clear pollutants in wastewater. One of the advantages is that biomass can
be retained on support under the working environment.

3.0 METHOD:
3.1 Preparation of fungal biomass
1. Phanerochaete chrysosporium was cultured at 32C for 7 days on potato dextrose agar
plates.
2. Spores on agar plates were scraped by distilled water to form a spore suspension.
3. About 5ml of spore suspension per 100 ml of culture medium containing malt extract and
activated carbon which is previously autoclaved at 121C for 15 minutes was added. The
culture medium without activated carbon was set as a control. About 0.1g of activated
carbon was used for preparation of fungal biomass.
4. The culture medium was incubated at 32C and 150 rpm for 2 days.
5. After 2 days, the mycelia balls were collected and were kept in oven at 80C for about 1
hour to prepare the fungal biomass.
6. The fungal biomass was ground into powder using a mortar and a pestle.
3.2 Preparation of dye solution
1. Stock solutions of methylene blue at different concentrations was prepared with distilled
water.
3.3 Batch biosorption studies
1. About 0.1g of fungal biomass powder with 50ml of methylene blue solutions at 20mg/l
and pH 10 were used.
2. The samples were shaken at 150rpm in incubator shaker at 30C for 60 minutes.
3. The samples were taken out from incubator shaker and were filtered to remove fungal
biomass.
4. The supernatant was analyzed to determine the concentrations of remaining methylene
blue in the solution using spectrophotometer set at a wavelength of 664 nm.
5. The concentrations of methylene blue in the supernatant were determined using a
standard curve.
5.0 DISCUSSION:
In this experiment, adsorption is used to remove the methylene blue dye in the aqueous solution.
Phanerochaete chrysosporium is used to immobilize activated carbon for bioabsorbent. Both the
fungal biomass and activated carbon are able to adsorb methylene blue from aqueous solution. In
this experiment, the effects of cell immobilization for methylene blue dye removal by
bioabsorbent are studied and are analyzed. Theoretically, there is a gradual increase in

bioadsorption with increasing contact time up to a certain length of time, the amount of dye
being adsorbed by activated carbon is not significant.
A standard curve for the methylene blue dye from the stock solutions was plotted by using the
axis of absorbance read at 664 nm versus dye concentration in mg/l. From the curve plotted, it
can be concluded that the higher the absorbance value, the higher the dye concentration is. This
is due to the higher the concentration of methylene blue, the higher the intensity of the colour
and hence the higher the absorbance value.
From the standard curve, a linear equation of y = 0.123 x + 0.345 was obtained. By using this
equation, the concentration of methylene blue in the solutions at any contact time (Ce) in mg/l
can be calculated as listed in Table 2 for both control and activated carbon. As the contact time
increases, the absorbance value as well as the concentration of dye in solution is decreasing. This
is due to bioadsorption. Bioadsorption is a process where bioabsorbents like activated carbon and
the fungal biomass adsorbs coloured substances (methylene blue) from the aqueous solution.
Since bioadsorption was taking place, the colour intensity of the solutions will decrease as the
time proceeds and hence there are decreases in absorbance value of the solutions as time
proceeds. By comparing the concentration of methylene blue in the solutions at any contact time
(Ce) in mg/l of control and PC activated with carbon, it can be noticed that the Ce for PC
activated with carbon is lower than that of control at the same contact time. This can be
concluded that the dye removal is done better and faster by the PC activated with carbon than the
control.
In addition, the removal efficiency, % of methylene blue dye by activated carbon immobilized
with fungal biomass increases as the contact time increased as shown in Table 3. This means that
the longer the contact time, the higher the removal efficiency. The results indicate that the
removal is higher at the beginning and this is probably due to the larger surface area of the
immobilized activated carbon being available at the beginning for the adsorption of methylene
blue.
The amount of adsorption at equilibrium (qe) can be calculated by using the equation below:
qe = (Co- Ce)V/W
From Table 4 and 5 which shows the amount of adsorption at equilibrium for PC activated with
carbon and control respectively, it can be concluded that the amount of adsorption at equilibrium
is increasing as the dye concentration in solution is decreasing.

Adsorption isotherms are mathematical models that describe the distribution of the adsorbate
species among liquid and adsorbent, based on a set of assumptions that are mainly related to the
heterogeneity/homogeneity of adsorbents, the type of coverage and possibility of interaction
between the adsorbate species. The equilibrium adsorption data are usually analyzed by
adsorption isotherms such as Langmuir and Freundlich isotherms. These isotherms relate the
amount of adsorption at equilibrium, qe with the concentration of methylene blue in solution, Ce.
The Freundlich isotherm is an empirical equation employed to describe the adsorption processes
on surface sites that are energetically heterogeneous while the Langmuir isotherm takes an
assumption that the adsorption occurs at specific homogeneous sites within the adsorbent. Graph
of ln qs vs Cs (Freundlich) and graph of Ce/qe vs Ce (Langmuir) for both PC activated with carbon
and control are plotted which are Figure 2, Figure 3, Figure 4 and Figure 5. From the graphs, it
can be concluded that in this experiment, Freundlich isotherm fitted well than the Langmuir
isotherm with a correlation coefficient of 0.664 for PC activated with carbon and 0.772 for
control.

6.0 CONCLUSION:
In conclusion, the higher the concentration of methylene blue, the higher the intensity of the
color and hence the higher the absorbance value. As the contact time increased, the
absorbance value as well as the concentration of dye in solution is decreasing due to
bioadsorption. The removal is higher at the beginning because there is a larger surface area of
the immobilized activated carbon being available at the beginning for the adsorption of
methylene blue. The PC activated with carbon have performed better dye removal task than
the control which do not have activated carbon since the dye concentration in PC activated
with carbon is lower than that of control at the same contact time. This means that PC
activated with carbon have removed higher concentration of methylene blue dye than that of
the control.

7.0 REFERENCE:
Ahmadpour, A., & Do, D. D. (1996). The preparation of active carbons from coal by
chemical and physical activation. Carbon, 34(4), 471-479.
Aksu, Z. (2005). Application of biosorption for the removal of organic pollutants: a review.
Process Biochemistry, 40(3), 997-1026.
Ferrero, F. (2010). Adsorption of Methylene Blue on magnesium silicate: Kinetics,
equilibria and comparison with other adsorbents. Journal of Environmental Sciences, 22(3),
467-473.
Gupta, V. K. (2009). Application of low-cost adsorbents for dye removalA review. Journal
of environmental management, 90(8), 2313-2342.
Hameed, B. H., Ahmad, A. L., & Latiff, K. N. A. (2007). Adsorption of basic dye
(methylene blue) onto activated carbon prepared from rattan sawdust. Dyes and Pigments,
75(1), 143-149.
Li, H., Li, Z., Liu, T., Xiao, X., Peng, Z., & Deng, L. (2008). A novel technology for
biosorption and recovery hexavalent chromium in wastewater by bio-functional magnetic
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