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Faculty of Engineering, University of Sumatera Utara No. 9 Jalan Dr. T. Mansur, 20155, Medan, Sumatera Utara, Indonesia*Corresponding Author: [email protected]
In many water bodies, water hyacinth has been a nuisance towards the ecosystem. Utilization of water hyacinth seems to be a promising way of controlling this weed. However, despite the amount of research on this particular matter, it is still not cost effective. Correct choice of process parameters and on0site enzyme production are believed to be able to cut the cost effectively. Therefore, this research aimed to evaluate and select the critical process parameters. In this research, pre0blended and filtrated water hyacinth was subjected to pretreatment. Afterwards, water hyacinth was hydrolysed and subsequently fermented and co0 fermented. Magnesium sulfate was added to enhance the process. Result favors the use of DAP as pretreatment, addition of inoculums for 24 h fermentation, and addition of magnesium sulfate in fermentation broth at 25 mM. Fermentation duration beyond 24 h led to decrease in sugar and ethanol, while addition of magnesium sulfate to level of 100 mM did not interrupt fermentation but caused underestimation in sugar analysis (DNS assay).
Keywords: Water hyacinth, Bioethanol, On0site enzyme, Pretreatment, Duration, Magnesium sulfate.
Water hyacinth has long been associated with negative socioeconomic and
environmental impacts [104]. Despite its water purifying ability [507], it still poses
significant hazard towards an ecosystem. Direct control of its growth has been
ineffective [104]. Hence, current research is driven towards converting the weed
to bio fuel as a mean of control [8, 9].
AN
CMC
Carboxymethyl Cellulose
CU
DAP
DNS
FPU
GB
LHW
PDA
SC
TR
Dilute Acid Pretreatment
Dinitrosalicylic acid
Filter Paper Unit
Liquid Hot Water
Potato Dextrose Agar
of enzymes; methods of hydrolysis; processing scheme; microbial choices;
and others [10025]. However, due to costly processing, the technologies
have yet to be applied. Correct choices of process parameters and on0site
enzyme production are considered important to realize the implementation of
the technology.
Therefore, this paper aimed to evaluate and select the critical process
parameters on bioethanol production from water hyacinth.
Water hyacinth was collected from local ponds in University of Sumatera Utara,
Medan, Indonesia.
(SC) and
(GB) were purchased from University of Sumatera Utara, Medan, Indonesia.
(TR),
(AN) and
(CU) were
purchased from Bandung Institute of Technology, Bandung, Indonesia. All
chemicals used were of analytical grade.
Water hyacinth was chopped into pieces and the roots were removed then, it was
blended to slurry and filter0pressed to reduce water content. A portion of the
filtered water hyacinth was dried further for analytical and experimental purpose.
Afterwards, both filtered and dried water hyacinth was analysed (procedure in
2.7) and stored in closed, separated container at 406 °C.
All microorganisms except SC, which was kept in granular form in closed
container at 8 °C, were grown in potato dextrose agar (PDA) at 20
oC. Prior to
usage, SC was warmed to 20
oC for 30 min, whilst TR, AN, and CU
were inoculated at 20
oC for 2 days (1 day for CU) in liquid media containing
22% sucrose, 1% KH
2PO
4, and 1% (NH
4)
2SO
4[26]. All procedures were
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Enzyme production was carried out in a 1000ml vial in which 10 g of water
hyacinth (moisture adjusted to 70%) was mixed with Mendel Weber solution at a
ratio of 3 ml to 1 g biomass. The mixture was autoclaved (121 °C, 15 lb) for 15
minutes, and subsequently cooled down to 20 °C. Inoculums of TR and AN (1.5
ml per 10 g biomass) were grown separately in prepared mixture and incubated at
20 °C for 1 week. Enzymes were extracted by distilled water at a ratio of 405 ml
per g biomass. The liquor was separated by centrifugation at 2.500 rpm and 4 °C
for 15 minutes. Supernatant from both cultures were then mixed at a ratio of 1:1
and stored in dark glass bottle at 4 °C.
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Water hyacinth was subjected to 4 types of pretreatments: simple sterilization,
dilute acid pretreatment (DAP), liquid hot water (LHW), and biological
pretreatment. In simple sterilization, 18 g of water hyacinth (94.4% moisture) was
mixed with 5 ml of distilled water, and autoclaved (121 °C, 15 lb) for 15 min. In
DAP, 1 g dried water hyacinth was mixed with 20 ml of 2% (v/v) sulfuric acid,
and autoclaved (121 °C, 15 lb) for 1 hour, followed by neutralization with
concentrated NaOH (5010 M) to pH of 405. In LHW, 18 g of water hyacinth
(94.4% moisture) was mixed with 5 ml of distilled water, and heated at 140 °C for
2 hours. In biological pretreatment, 0.5 g of the white rot fungus (GB with PDA
included) was added to 18 g of water hyacinth (94.4% moisture) and incubated
for 7 days at 20 °C. After incubation, 5 ml of distilled water was added. After
each pretreatment, all samples were cooled down to 20 °C. All the types and
conditions of pretreatments were adjusted from results reported on various
journals [13, 16020, 22025].
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The hydrolysis and fermentation were carried out simultaneously. After
pretreatment, 30 ml enzymes, 001.23 g (000.1 M) MgSO
4, and 0.5 g (1% w/v) SC
were added, and whole mixture was incubated at 20 °C for 24096 h. As
alternative, 1 ml (2% v/v) CU inoculums were added at the beginning of
fermentation or after 24 h fermentation. After incubation, fermentation broth was
filtered and the filtrate was analysed. A high enzyme volume was required due to
low enzyme activity but was kept at low enough volume to avoid excessive
dilution. Concentration of MgSO
4was kept in range of 00100 mM to observe the
effects on fermentation at higher concentration as studies on this subject suggest
that MgSO
4has positive effect at concentration of 3.5 mM but becomes inhibitory
at higher concentration [27028]. Influence of various parameters on hydrolysis
and fermentation was optimized by step0wise experiments where specified
parameter was changed by keeping all other parameters constant. Most effective
parameter was selected for further optimization of process parameters.
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analysed by Chesson0Datta method [29]. Crude enzyme was analysed for its
cellulase activity by CMC assay and the activity was expressed as FPU/ml [30].
(
Concentration of reducing sugar was analysed by spectrophotometer UV0Visible
(SHIMADZU 1800) using DNS method [31] and was expressed as equivalent
glucose concentration against calibration curve. Ethanol concentration was
analysed by GC using static head space analysis [32] at adjusted salt
concentration of 0.1 mM MgSO
4against calibration curve. Iso0propanol was used
as an internal standard. Density, viscosity, and pH were measured by using
pycnometer, Oswald viscometer, and pH meter.
)
*
Moisture content analysis on chopped water hyacinth showed that the stems
contain 92.1 0.3 % of water, while the leaves contain 87.0 0.7 % of water.
Mixed, blended and filtered water hyacinth contains 94.4 ± 0.1 % water. Results
of chemical content analysis, along with results from other studies, are shown in
Table 1. Crude enzyme was found to have cellulase activity of 0.12 FPU/ml.
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Pretreatment affects hydrolysis of cellulose by modifying lignocellulose structure
to allow for better enzyme access [33035]. Effectiveness of pretreatments depends
on type of biomass because of differences in structure and composition. Results
on effect of pretreatments are shown on Fig. 1.
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Magnesium sulfate increases yeast tolerance on alcohol by decreasing cell
membrane permeability [27]. Salts in general also induce salting out effect on water
– organic system, in which most salts alter equilibrium dynamic of both phases and
drive ethanol out of water [36, 37]. In this study, effect of magnesium sulfate was
studied by varying the magnesium concentration in the range of 00100 mM
(001.23 g). Magnesium sulfate was added after DAP and neutralization, and the
salt concentration was adjusted to 100 mM before sugar and ethanol analysis.
As shown in Fig. 2, magnesium concentration did not have detrimental effect
on fermentation, although at 25 mM, sugar and ethanol concentration were
slightly higher than those at other magnesium concentration.
A higher MgSO
4concentration could not be tested because the salt disturbs
sugar analysis by DNS method as shown in Fig. 3. The existence of Mg
2+ions are
also reported to interfere with the activity of cellulase, although further
verification is required to determine whether the cause of the decline in enzyme
activity is due to direct inhibition of the ions on cellulase, or disturbance on the
enzyme assay [38].
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Duration affects the composition and microenvironment of the broth. In ethanol
fermentation, an overlong duration may result in ethanol loss by decomposition,
evaporation, and conversion to other products [39], while insufficient duration
will lead to lots of unconverted sugar and lower ethanol yield. In order to
optimize the duration, a duration range of 24096 h was tested. The results are
shown in Fig. 4.
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Results agree well with the literature in which longer fermentation did not
lead to more ethanol even though sugar was consumed. In some literatures,
ethanol fermentations with quite similar conditions required less than one day
fermentation [11, 24].
0.0 0.3 0.6 0.9 1.2
0 1 2 3 4
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Microorganism affects the ethanol yield. Microorganisms of same species could
give different yield depends on the strain. If the microorganisms are of different
species, they will require different fermentation conditions. In this study, SC was
chosen for hexose fermentation and CU for pentose fermentation. Results are
shown in Fig. 5.
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Results indicate that CU contributed positively on ethanol production.
Sornvoraweat and Kongkiattikajorn (2010) acquired similar results by using co0
culture of
and
.
The increase on ethanol concentration could be attributed to different substrate
utilization by both yeasts. However, it is also possible that extra sucrose from
inoculums increased the ethanol yield as well. Due to the many factors which
influence the performance of both yeasts, such as differences in magnesium
absorption and accumulation in both yeasts which change dynamically [40], glucose
limited competition between both yeasts which depends on the aerobic or anaerobic
state [41], complicated ethanol generating system of
which requires
auto anaerobic setting along with ethanol repressing system which activates on
stationer phase [42], a more focused and thorough research is required to understand
the real cause of the increase of ethanol yield in this system.
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Effects of pretreatment, magnesium sulphate, fermentation duration, and microbial
choices on bioethanol production from water hyacinth were investigated. Some
conclusions are given below.
For bioethanol production from the cellulose portion of water hyacinth, DAP
is still the best pretreatment despite reported inhibitor generation.
0 1 2 3
A B C
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In the range tested, magnesium salt does not have significant effect on sugar
and ethanol yield.
Fermentation duration of one day was sufficient for maximum yield.
Adding CU also yielded positive result for 1 day fermentation.
In all the experiments, ethanol and sugar concentration were not high. This
might be due to the low enzyme loading. Further addition of enzyme
mixture is possible but not initiated because extra cost for ethanol
purification quickly override the advantage of cost cutting effect given by
on0site enzyme production.
Although enzyme production from merely dried0blended water hyacinth is
possible, it could not achieve the goal to cut the cost of bioethanol production
in this research. Thus, another approach has to be taken. Some possible
approaches may include altering water hyacinth composition to better suit
cellulase production, or eliminating extraction step by direct mixing of
incubated biomass. The latter choice suffers from possibility of competitive
substrate utilization and product assimilation. Composition alteration can be
achieved by treating water hyacinth by DAP and taking the solid portion, as
pretreated water hyacinth will contain higher cellulose and reflects substrate
composition in ethanol production better.
Further research on bioethanol from water hyacinth should investigate other
strategies to cut production cost. The strategies may include study on
purification step as it is considered one of the most cost and energy
consuming steps in general, utilization of fermentation waste for further
economic value, and increasing ethanol yield either by improved yeast strain
or better processing strategy.
2
The authors would like to thank Indonesia Endowment Fund for Education (LPDP)
under Ministry of Finance, Republic of Indonesia for funding the research.
)
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