BIOCATALYSIS FOR BIODIESEL
E.T. Lau, M. Xiao, J. Tan
Environmental Science and Engineering
National University of Singapore
21 Lower Kent Ridge Road, Singapore 119077
ABSTRACT
The extent of biocatalysis, using a local fungal isolate enzyme for conversion of a range of PDOs into biodiesel, was studied. Ratio
concentrations of 0.25, 0.5, 0.75 and 1.0 of Tert Buthanol to methanol were used for the biocatalysis process. On the whole, the results
obtained were unsatisfactory due to the low yield of biofuel attained (ME%) at the end of the experiment. Freeze-dried beads and nondried beads of 72 hr were tested for their yield amount, however the results showed insignificant differences. It was also noted that the
beads that did not have the addition of buffer obtained higher ME% readings. Further tests concluded that the addition of buffer could
have inhibited the reaction to a certain extent, thus resulting in lower ME%. Lastly, hypothesis was made that there may be a critical
ratio concentration where good conversion results can be achieved, other than that, the amount obtained will be low. Therefore, this
experiment would need further investigation works and analysis.
1.
INTRODUCTION
1.1 Background
A huge amount of waste edible oils are discharged yearly. Half of this portion is estimated to be recycled as animal feed or raw
materials for lubricant and paint. The remainder, however, is discharged into the environment. Hence, production of a biodiesel fuel
(fatty acid methyl esters, FAMEs) from waste edible oil is considered an important step of reducing and recycling waste oil1.
Presently, the industrial production of biodiesel fuel is performed by methanolysis of waste oil using alkaline catalysts. A by-product,
glycerol, containing the alkali, has to be treated as a waste material. In addition, because waste oils contain a small amount of water
and free fatty acids (FFAs), the reaction generates fatty acid alkaline salts (soaps). The soaps are removed by washing water, which
also removes glycerol, methanol (MeOH), and catalyst. Hence, disposal of the resulting alkaline water creates other environmental
concerns. On the other hand, since enzymatic methanolysis of waste oil does not generate any waste materials, production of biodiesel
fuel with lipase is strongly desired. Several studies have reported the utilization of microorganisms such as bacteria, yeast and fungi as
whole-cell biocatalysts in attempts to improve the cost effectiveness of the bioconversion processes. Among the established wholecell biocatalyst systems, filamentous fungi have arisen as the most robust whole-cell biocatalyst for industrial applications2. This
report will cover the experiment of whole cell biocatalysis using lipase producing fungal strains to convert plant derived oils (PDOs)
into biodiesel via chemical transesterification, in an engineered bioreactor. A local fungal isolate will be used for the biocatalysis
process.
1.2 Objectives
To investigate the extent of biocatalysis, using the lipase enzyme for conversion of a range of PDOs into biodiesel.
2.
MATERIALS AND METHODS
2.1 Ceramic beads immobilised biocatalyst preparation
2.1.1. Microorganisms and Medium The materials needed for Basal medium, for the cultivation of fungi sample, are 70g/L Polypeptone, 1.0g/L Sodium Nitrate, 1.0g/L
KH2PO4, 0.5g/L Magnesium Sulphate, 3% Olive oil (30g/L), amounting to 100 ml per flask. The prepared medium is then stabilized
by autoclave.
After 7 days’s cultivation, fungus is cut into 10mm square cubes and put into the Basal medium flasks for inoculation to take place.
The fungi mixture is incubated for 6 days, at 25⁰C in an Orbital Incubator shaker at 150rpm. Once inoculation is done, biomass is
extracted by washing off the Basal medium, from whole cell fungi, 3 times using a vacuum filter.
2.1.2. Preparing ceramic beads: Two Percent of Sodium Argenate is mixed with each of the freeze-dried biomass and non-freeze dried biomass. The mixture is then
placed through a stringe, attached to a pump, and droplets are slowly formed and solidified once dropped into the solution of Calcium
Chloride (2%). This entrapped solidified biomass is known as Calcium Argenate (enzyme beads) which is then used in the biocatalysis
process.
2.2 Methanolysis in solvent with BSPs immobilised biocatalysts 1
Step 2.1.1 is done. After 6 days of incubation, ceramic beads are harvested by separating the culture from the medium broth and
washing with tap water for 5 times. Then it is freeze-dried for 24 hours and kept in the desiccator.
2.3 Methanolysis reaction
Stock sample is prepared by using the following reagents: 10g Palm oil and 1.14g Methanol. Tert Butanol Solvent is then added to the
mixture of Palm oil and Methanol (‘A’) in the ratio as shown from table 1 below.
Sample no.
1
2
3
4
5
6
7
8
9
‘A’
1 1 1 1 1 1 1 1 1 Tert Butanol
0.25 0.25 0.5 0.5 0.75 0.75 1 1 1 In addition, after ratio mixing, 0.5ml of pH 6.5 Sodium Phosphate buffer and 40 pieces of enzymes are added to each flask.
Methanolysis is then carried out in a 50ml screw-capped bottle at 40⁰C in an Orbital Incubator shaker at 150rpm for 3 durations: 24 hr,
48 hr and 72 hr.
The conical tray of extracted samples are then stored in the fridge till the next extraction at 48 hr. Then at 48 hr from the start of
methanolysis reaction, another 9 samples are extracted the same way as explained above for the 24 hr samples. The steps are then
repeated for the 72 hr samples.
After which all 27 samples are extracted on the conical tray, all the caps of the conical holders are opened and the tray is placed into
the oven to be heated at 70⁰C for 2 hours.
Next, the 24hr samples are weighed after heating and then placed in the centrifuge, set to 25⁰C, 12,000 x g, for 5 minutes. (Making
sure the placement of the conical holders, in the centrifuge, is balanced). Then, the centrifuged samples are diluted by 25,000 times,
where the following mix below states the amount of dilution done.
Mix 1: 100x dilution- take 100uL from samples to make 1ml.
Mix 2: 50 x dilution- take 20uL from Mix 1 to make 1ml.
Mix 3: 50 x dilution- take 20 uL from Mix 2 to make 1ml.
The centrifugation and dilution procedures explained above are repeated for both the 48hr and 72hr batches.
2.4 Analysis
The diluted samples are then injected into a QP2010 gas chromatograph mass spectrometer (GCMS) and analysed batch by batch. In
another words, the 24hr, 48 hr and 72 hr batches are analysed one after another.
3.
RESULTS AND DISCUSSION
3.1 Methanolysis with Ceramic beads immobilised biocatalyst
3.1.1 Dried and Non­dried 72 hr samples Generally, the differences are insignificant for freeze-dried and non-dried beads in terms of the ME% achieved. However, comparing
within the non-dried beads, higher ME% is achieved for beads that did not have the addition of buffer. This outcome could be caused
by the fact that the buffer may have inhibited the reaction slightly, thus resulting in lower yield (ME%) amount.
Comparing within the dried beads, the sample with lower concentration of palm oil unexpectedly achieved a higher yield as compared
to the sample with higher concentration of palm oil. This result should not be the case as it is theoretically incorrect and may be due to
the accumulation of errors during the experiment. From figure 1, we note that the standard deviation for the low concentration dried
beads is relatively high, thus it justifies the existence of possible errors that may have occurred when carrying out the experiment.
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Figure 1
3.1.2 Varying sized and percentage buffered 72 hr samples Figure 1
Sample 1: big beads with 5% buffer
Sample 2: big beads with 8% buffer
Sample 3: big beads buffer rinse with 5% buffer
Sample 4: big beads buffer rinse with 8% buffer
Sample 5: small beads buffer rinse with 5% buffer
Sample 6: small beads buffer rinse with 8% buffer
Extracting only the new 72 hr samples, the level of ME% achieved for the different types of samples, in increasing percentages, are as
follows: (i) big beads buffer rinse with buffer, (ii) small beads buffer rinse with buffer and lastly (iii) big beads with buffer. The higher
ME% achieved by (i) as compared to (ii) may be due to the fact that the smaller beads have a larger surface area, thus in contact with a
higher amount of buffer, therefore is inhibited more. This supports the findings we have in 3.1 where higher buffer content could have
possibly inhibited the reaction and resulted in a lower ME% achieved.
Comparing (i) and (ii), the samples which were rinsed with buffer registered a higher ME%. In addition, comparing the results from
using different percentages of buffer, the beads with 5% buffer obtained a higher yield than beads with 8% buffer. These 2 findings
further substantiate the explanation for 3.1 and above.
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3.2 Methanolysis in solvent with BSPs immobilised biocatalysts
Time course of ME Yield
Figure 3 Generally, results obtained were unsatisfactory due to the low yield of biofuel attained at the end of the
experiment. This can be noted from the small numbers shown in Table 3, under “Average ME%”.
The insignificance of the biofuel yield (ME%) is an indication of low enzymatic activity. This could have been
the result of many factors such as (i) the enzyme added could not mix properly with the solvent and (ii) the
mixture of oil with methanol could have been non-uniform as oil is immiscible in methanol. To minimize the
errors incurred from factor (ii), we used Tert Butanol to solubilize the reagents so that the mixture of oil,
methanol and enzyme will be better mixed and solution will be more uniform.
The low biofuel yield could also have arisen from the fact that Methanol will inhibit enzymatic reactions at
usage of more than 1.5 moles. As triglyceride exists in palm oil compound, 3 moles of methanol is needed for
the process of methanolysis. Thus the overusage of methanol is most likely going to inhibit the enzymatic
reaction taking place. To overcome this problem, we added 1 mole of methanol at one time, thrice, into the
solvent system.
From Figure 3, large inconsistencies are noted. There is no general pattern observed for the 4 ratio
concentrations used and also in regards to the duration where the enzymatic reactions are left to take place.
However, only the graphical data of ratio concentrations 0.25 seems to agree theoretically as the ME% increases
with the time when the enzymatic reactions are left to take place. As for the remaining 3 ratio concentrations, I
believe that the unsatisfactory results stem from numerous experimental errors.
The improvement to this experiment could be finding a critical ratio concentration. This time, only ratio
concentrations of 0.25, 0.5, 0.75 and 1.0 were used and the results were not very satisfactory. Thus, I
hypothesize that there may be a critical ratio concentration where good conversion results can be achieved, other
than that, the amount obtained will be low. Therefore, this experiment would need further investigation works
and analysis.
4.
CONCLUSION
Through this experiment, insignificant yield were obtained through different operational controls of the enzyme
beads. Thus, we can conclude that the extent of biocatalysis, using a local fungal isolate enzyme for conversion
of a range of PDOs into biodiesel, was unsatisfactory. Though whole-cell biocatalysis can provide further
advantages over conventional enzymatic processes with regard to providing an inexpensive method of catalyst
preparation and excellent operational stability, further improvement in the reaction rate of whole-cell catalyzed
biodiesel fuel production will be necessary for practical industrial applications.
5.
1.
2.
REFERENCES
F.R. Ma, M.A. Hanna, Biodiesel production: a review, Bioresour. Technol. 70 (1999) 1-15.
H. Fukuda, S. Hama, S. Tamalampudi, H. Noda, Whole-cell biocatalysts for biodiesel fuel production: a
review, ScienceDirect.
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