EnCon 2012, 5th Engineering Conference, "Engineering Towards Change - Empowering Green

Solutions" 10-12th July 2012, Kuching Sarawak

Optimization of Bacterial Cellulose Production from

Pineapple Waste: Effect of Temperature, pH and
Concentration
Junaidi Zakaria and MuhammadAzlan Nazeri
Faculty of Chemical & Natural Resources Engineering
Universiti Malaysia Pahang, Lebuhraya Tun Razak,26300, Kuantan, Pahang
Email: [email protected], [email protected]

Abstract

Bacterial cellulose is a type of biopolymer produced by Acetobacter xylinum in high purity, high water holding capacity, good
mechanical strength, elasticity and high crystallinity. In this research, pineapple waste is used as the carbon sources for the synthesis of
bacterial cellulose. The objective of this study is to investigate the effect of temperature, pH and concentration of pineapple waste in the
production of bacterial cellulose by Acetobacter xylinum. Parameters investigated are varied from 40% to 100% for the concentration,
while the temperature is between 28C to 32C and pH of 4.5 to 8.5. Besides, this study also aims to optimize the production of bacterial
cellulose from pineapple waste by using response surface methodology (RSM) based on the central composite design (CCD). The known
value of the parameters is estimated earlier based on one factor at that time (OFAT). The results obtained from the OFAT showed the
optimum condition is at pH 5.50, temperature 30C and concentration of pineapple waste is 80 %, where the amount of bacterial cellulose
dry weight is 3.3948g. According to the RSM result, the optimal conditions for bacterial cellulose were pH 5.15, temperature 30.51C and
concentration of pineapple waste is 83.32%. By using these optimal conditions, 3.4368g of bacterial cellulose is produced. The existence
of bacterial cellulose is proven by Fourier Transform Infrared (FT-IR) Spectroscopy analysis based on the appearance of absorbance peak
which are C-C bonding, C-O bonding, C-OH bonding and C-O-C bonding. In short, the data presented in this paper showed that pineapple
waste has a great potential to use as the carbon source in production of bacterial cellulose

Keywords: bacterial cellulose, Acetobacter xylinum, pineapple waste

I. INTRODUCTION

Cellulose is the most abundance polymer present as the major component of plant biomass and a representative of
microbial extracellular polymers. Acetobacter xylinum is the bacteria that able to grow on the waste material to produce
cellulose. The several genera that have shown the ability to synthesize cellulose include Sarcina, Agrobacterium, Rhizobium
and Acetobacter (Barbara et al., 2008). However species that able to produces cellulose in high quantity is Acetobacter
xylinum. Over a century ago, this organism and its produce were first identified and characterized (Iguchi and Yamanaka,
1997).
Bacterial cellulose is a polymer produced by Acetobacter xylinum in presence of glucose. Bacterial cellulose has high
purity cellulose where it free from lignin and hemicelluloses differ from plant cellulose that has low purity cellulose and
containing lignin and hemicelluloses (Klemn et al., 2001). There are several aspect that differentiate bacterial cellulose with
plant cellulose which are bacterial cellulose has unique characteristic including good mechanical strength, high water
absorption capacity, high crystallinity, ultra-fine and highly pure fibre network structure that caused bacterial cellulose is
preferred than plant cellulose (Keshk and Sameshima, 2006). There are four different pathways in forming the cellulose
biopolymer (Klemn et al., 2001). The first pathway is by the isolation of cellulose from plants. This pathway involved
another separation process step to remove lignin and hemicelluloses. The second pathway is the synthesis of cellulose by
microorganism such as Acetobacter xylinum. In the synthesis process, bacteria that can produce highest cellulose amount is
Acetobacter xylinum. It produced cellulose in the form of extracellular pellicle composed of ribbons, Achromobacter,
Aerobacter, Alcaligenes produce cellulose in fibrils form while Agrobacterium and Rhizobium produces cellulose in the form
of short fibril (Barbara et al., 2008). The third and fourth method are by the first enzymatic in-vitro synthesis starting from
cellobiosyl fluoride and the first chemosynthesis from glucose by ring opening polymerization of benzylated and
pivaloylated derivatives (Klemn et al., 2001)
Cellulose is the main part in the cell wall plant and act as protective and coating, whereas plant cellulose plays a structural
role in plant (Bielecki et al 2000). However cellulose is obtained from the plant is not pure because it have lignin and
hemicellulose, so it is difficult to purify the cellulose from them through separation process. Nowadays bacterial cellulose
 EnCon 2012, 5th Engineering Conference, "Engineering Towards Change - Empowering Green Solutions" 10-12th July
2012, Kuching Sarawak
used as an alternative instead of plant cellulose in order to produce high purity cellulose and in the same time to reduce the
forest depletion (Sherif, 2008). Most of the paper production used cellulose pulp from plant and thus gives a problem on
forest depletion and now many research has been conducted on producing paper from bacterial cellulose and as a result,
there is an improvement of the papers strength properties and protect the surface of paper (Barbara et al., 2008). The form
of its size, crystallinity and purity had differentiated between bacterial cellulose produced by bacteria that has a unique
physical and chemical properties with cellulose that produced from plant (Prashant et al., 2008).
Bacterial cellulose also has disadvantages that need to encounter although bacterial cellulose has a unique characteristic
than the plant cellulose. The main problem is the price for the sugar as a substrate is very expensive but low in production.
Using the fruit waste such as fruit peel is one of the alternatives that can overcome this problem. The mango peel, pineapple
core, watermelon peel and other fruit wastes are the example of fruit waste that can be utilizes as substrates to produce
cellulose (Akihiro et al., 2008).

2. METHODOLOGY

2.1 Experimental Procedures

2.1.1 Preparation of Inoculum

250mL of conical flask is filled with 200mL of deionized water, 4g glucose, 1g peptone, 1g yeast extract, 0.54g disodium
phosphate and 0.23g citric acid. Then, it is sterilized in autoclave. After that, 20mL stock culture of Acetobacter Xylinum sp
is added into the conical flask. The culture broth was incubated at 30C for 3 days. After 3 days, the inoculums is ready for
the next fermentation process

2.1.2 Preparation of Pineapple Waste Juice

300g of pineapple residue is weighted. The pineapple residue collected was crushed and blend with 200 mL of water using
blender. After that, pineapple residue was filtered with a filter cloth to separate the pineapple waste juice and then it was
sterilized using an autoclave.

2.1.3 Synthesis of Bacterial Cellulose

250mL conical flask is filled with 100mL of fermentation medium consist of 60mL of distilled water, 4g of yeast, 45 g of
peptone, 1.035g of citric acid, 2.43g of disodium phosphate and 0.45g of magnesium sulphate. 40mL of pineapple waste
juice with concentration of 40% is added to the medium. Then, the fermentation medium is sterilized by using autoclave.
After that, 10mL of the inoculums is transferred to the medium. The medium was incubated at 30C for 5 days. After 5 days
of incubation, the pellicles formed at the surface layer are treated with 1% sodium hydroxide for 30 minutes at temperature
90C then it is rained with distilled water. Lastly, weight of the pellicles is measured. All the steps are repeated for different
incubation temperature of 28C,29C,30C,31C,32C, pH medium of 4.5,5.5,6.5,7.5,8.5 and concentration of pineapple
waste juice of 40%, 50%, 60%, 70%, 80%, 90% and 100% .

2.1.4 Bacterial Cellulose Analysis

The cleaned bacterial cellulose is dried at 60C for 5 hour to remove the water content. After drying for 5 hours, the dried
bacterial cellulose is analyzed using FTIR. After that, the graph for FTIR is collected and studied.

3.0 RESULTS & DISCUSSION

3.1 Bacterial Cellulose Analysis

One of the most useful method to identify a chemical compounds based on the absorption of radioactive by the
compounds chemical bonds is the Fourier Transform Infrared Spectroscopy. C-OH bonding, anti-symmetric bridge
stretching of COC, C-C stretching at C6 (Sun et al., 2008), H bond in OH group, aliphatic OH group (Guo et al., 2008),
C-O stretching at C3 and C-O stretching are the chemical bonding that present in cellulose molecular structure. Figure 3.1
show the absorbance peak at 3744.97cm-1, 3615.08cm-1, 3410.62cm-1, 3332.89cm-1 and 3114.28cm-1 that is originated from
the OH stretching. This result had been proven from the previous study by Parmjit, et al., 2008 where the peaks that appears
near in range of 3853 -3256 cm-1 is hydroxyl functional group. The absorbance peak at 1393.77cm-1 is also showed and
according to the Sun et al., 2008, several bands typical for bacterial cellulose were shown in region of 1500-1235cm-1 due to
in plane bending vibration of CH2, CH, OH groups. In addition, absorbance peak at 1393.77 cm-1 also represent the strong
bond in bacterial cellulose due to C-O symmetric stretching (Esin et al., 2011). From the figure, absorbance peak also
 EnCon 2012, 5th Engineering Conference, "Engineering Towards Change - Empowering Green Solutions" 10-12th July
2012, Kuching Sarawak
appeared at 1076.39 cm-1. The strong band in bacterial cellulose that appears near 1081cm-1 is representative for the C-O-C
asymmetric stretching (Sun et al., 2008). Besides, there is also absorbance that appears at 1612.90cm-1. This absorbance peak
attributing to the bending mode of the absorbed water in the cellulose.

Figure 3.1: FT-IR spectra for bacterial cellulose (cm-1)

3.2 Synthesis of Bacterial Cellulose

3.2.1 Effect of Temperature

4
Dry Weight of Bacterial

3.5 3.3948
3
Cellulose (g)

2.5
2
constant of pH 5.5 and
1.5
1.156 temperature 30C
1
0.615
0.5 0.369
0 0.0957
27 28 29 30 31 32 33
Temperature(C)

Figure 3.2: The effect of temperature

3.2.2 Effect of pH medium

4
Dry Weight of Bacterial

3.5 3.3948
3
Cellulose(g)

2.5
2 2.1055
constant concentration 80%
1.5
1.0837 1.183 and temperature 30C
1
0.5 0.4886
0
0 2 4 6 8
pH Medium

Figure 3.3: The effect of pH

 EnCon 2012, 5th Engineering Conference, "Engineering Towards Change - Empowering Green Solutions" 10-12th July
2012, Kuching Sarawak
3.2.3 Effect of Pineapple Waste Concentration

4
3.3948

Dry Weight of Bacterial

3.5
3 2.9955
3.0257 2.7792

Cellulose (g)
2.5
2 2.5057
2.1772 1.6572 constant ph 5.5 and
1.5
1 temperature 30C
0.5
0
0 50 100 150
Concentration Medium (%)

Figure 3.4: The effect of concentration of pineapple waste

Results from OFAT showed that the optimum temperature is 30C, pH of 5.5 and concentration of pineapple waste is 80%.
Thus, these factors is used for optimization study to obtain higher yield of bacterial cellulose by using RSM.

Table 3.1: Annova for response surface quadratic model for the production of bacterial cellulose

Sources Sum of Degree Mean F- P-Value R2

square of square value (Prob>
freedom F)
Model 27.84 9 3.09 64.45 <0.0001 0.9831 Significant
A-pH 4.17 1 4.17 86.89 <0.0001
B-Temperature 0.90 1 0.90 18.69 0.0015

C- 0.14 1 0.14 2.85 0.1226

Concentration
medium
A2 6.10 1 6.10 127.13 <0.0001
B2 8.74 1 8.74 182.11 <0.0001
C2 10.62 1 10.62 221.27 <0.0001
Residual 0.48 10 0.048
Lack of Fit 0.29 5 0.059 1.59 1.59 Not significant
Pure Error 0.19 5 0.037
Correlation 28.32 19
Total

Table 3.1 recorded that the regressions for dry weight of bacterial cellulose were significant at <0.0001 and those lacked
of fit was not significant at P is 1.59 and the values are greater than 0.1000 that indicate the model terms are not significant.
The fits of the model were checked by the correlation coefficient, R2. The R2 value provides a measure of how much
variability in the observed response values can be explained by the experimental factors and their interactions. The R2 value
always lied between 0 and 1. The closer R2 value to 1.00, the stronger the model was and the better it predicted the response.
In this case, the R2 value for dry weight of bacterial cellulose is 0.9831. These values showed that 31.22% of the total
variable were not explained by the model. The Pred R2 of 0.9040 for dry weight of bacterial cellulose was reasonable
agreement with Adj R2 of 0.9678. This indicated a good agreement between the experimental and predicted values for dry
weight of bacterial cellulose. The adjusted R2 corrected the R2 value for the sample size and for the number of terms in the
model. If there are many terms in the model and the sample size is not very large, the adjusted R2 may be noticeable smaller
than R2. This should be the caution signal as too many terms were present in the model. The plot of predicted versus
experimental dry weight of bacterial cellulose are shown in Figure 3.5 with R2=0.9831, thus indicating an excellent adequacy
of the proposed model. In Table 3.1, the P-value obtained for regression model <0.0001 compared to a desired significant
level of 0.05. This signified that the regression model is precise in predicting the pattern of significance to the production of
bacterial cellulose.
 EnCon 2012, 5th Engineering Conference, "Engineering Towards Change - Empowering Green Solutions" 10-12
10 th July
2012, Kuching Sarawak

Figure 3.5: Plot of predicted versus experimental data for dry weight of Bacterial Cellulose.

In order to investigate the effects of the parameter

parameters on the dry weight of bacterial cellulose, the response surface
methodology is used and the tree-dimensionalional plot is drawn. Figure 3.63.6, 3.7 and 3.8 shows the response surface plot for the
parameters studied. Temperature, pH and concentration of pineapple waste medium gave the significant effect to the
production of bacteriall cellulose. From the figure 3.6, 3.7 and 3.83.8, the result indicated within the range of 5.25-5.75
5.25 for pH,
temperature of 29.5C -30.5 C and 75%-85% 85% of concentration, the yield of bacterial cellulose is increased.
increased After this range,
the production is slightly decreased. The maximum production of 3.48818g of bacterial cellulose is obtained when pH of
5.15, 30.51C of temperature and 83.32% of concentration is used.
Based on report by Chawla et al., 2008, Acetobacter xylinum is active to synthesize bacterial cellulose during acidic
condition because it is a type of acetic acid microbe
icrobe that needs acidic condition for growth. The data indicated that as the pH
outside from the optimum pH range 5.25-5.75 5.75 resulted decrease in bacterial cellulose production.. This showed that more
gluconic acid is formed in range of pH 5.00-5.25.
5.25. According to the Klemn et al., ., 2001, during cultivation, gluconic acid and
5-keto-gluconic
gluconic acid are responsible for the decrease of the pH value and gluconic acid is not beneficial for overall bacterial
cellulose productivity.
Based on report by Jonas and Farah, 1998 the optimal growth temperature for bacterial cellulose production is 25C -
30C, although most researchers observed that 30OC is the best temperature for cellulose yield. Figure 3.6 and 3.8 revealed
that the highest value of bacterial
terial cellulose yield is at temperature of 29.50C -30.50C and the optimum temperature is
30C. According to the report by Chawla et al 2008, the conversion of glucose to cellulose is regulated by a multi step
carbon metabolism pathway involving a large number of both individual enzymes and complexes of catalytic and regulatory
proteins. For the temperature range 30.50C to 31 31C, the bacterial cellulose yield is decreased because of the enzyme cannot
perform well when the temperature is overr than optimum temperature. Suitable temperature is between 28C to 30C for the
enzyme to work at optimum performance.
In the synthesis of bacterial cellulose, Acetobacter xylinum used the natural glucose present in the pineapple waste as
carbon source. In the normal condition, Acetobacter xylinum produced thicker layer of bacterial al cellulose by synthesizing
more glucose resulting to higher yield of bacterial
acterial cellulose as shown in FFigure 3.7 and 3.8.. For other case, the data indicated
that as the concentration outside the optimum concentration range 75% 75%-85%, resulted decrease in bacteria cellulose
production due more gluconic acetic or lactic acid formed when more glucose is synthesized during fermentation process.
This side product will accumulate into the medium and ddecreases the pH level, thus decrease the bacterial cellulose
production (Prashnant et al., 2008)

Figure 3.6: Temperature vs pH Figure 3.7:: Concentration vs pH

 EnCon 2012, 5th Engineering Conference, "Engineering Towards Change - Empowering Green Solutions" 10-12
10 th July
2012, Kuching Sarawak

Figure 3.
3.8: Concentration vs temperature.

3.3 Validation of the Model

The production of bacterial cellulose

lose was successfully optimized and Table 3.2 shows the summary of the optimized
conditions. In order to validate the adequacy of the model, a total of three verifications
erifications experiments for wet weight of
bacterial cellulose responses are carried out under various fermentatio
fermentation conditions as shown in Table
able 3.2.
3.2 The verification of
the results was accomplished by carrying out the experiments under optimal condition of pH 5.15, temperature of 30.51oC
and concentration of 83.32%. The agreement reached between the predicted and experimental result verifies the validity of
the model and existence of an optimal point with error 0.0147
0.0147.

Table 3.2 Validation

ion of the data and models constructed for Bacterial Cellulose yield.

Condition After Optimization Before Optimization

Value Wet weight Bacterial cellulose (g) Value Wet weight of
bacterial
cellulose (g)
pH 5.15 Predict Experimental 6.00

Temperature (C) 30.51 31

3.48818 3.4368 1.4703
Concentration (%) 83.32 90.00

Error () = 0.0147

4.0 CONCLUSION AND RECOMMENDATION

As conclusion, the results obtained showed the optimum bacterial cellulose production is at temperature of 30.51oC, pH of
5.15 and concentration of pineapple waste is 83.32% where the amount of bacterial cellulose produced is 3.4368g.
Temperature, pH and concentration off pineapple used in this research are major parameters that gave ve major effect in the
synthesis of bacterial cellulose. Besides that, this result also proved that pineapple waste that high in glucose content has
great potential to be used for the synthesis
ynthesis of bacterial cellulose. Future works to scale up the production of bacterial
cellulose are highly recommended.

5.0 REFERENCES

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Barbara Surma-Slusarska,
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Bielecki, P. D., Krystynowicz, D. E., Mariannaturkiewicz, P. D., & Kalinowska, D. E. (2000). Bacterial Cellulose, 40
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Chawla, P. R., Bajaj, I. B., Survase, S. A.,, & Singhal, R. S. (2008). Microbial Cellulose: Fermentative Production And Application. Food Technology And
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Iguchi, M., and Yamanaka, S. (1997). Industrial use of bacterial cellulose. A review. Proceedings of International Workshop Green Polymer. Bandung
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ACKNOWLEDGEMENT

Financial support from Universiti Malaysia Pahang is highly appreciated.