PROCESS SELECTION ON BIOETHANOL PRODUCTION FROM WATER HYACINTH (EICHHORNIA CRASSIPES) OKTA BANI, TASLIM, IRVAN, IRIANY Faculty of Engineering, University of Sumatera Utara No. 9 Jalan Dr. T. Mansur, 20155, Medan, Sumatera Utara, Indonesia Corresponding Au

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Faculty of Engineering, University of Sumatera Utara

No. 9 Jalan Dr. T. Mansur, 20155, Medan, Sumatera Utara, Indonesia

• *Corresponding Author: taslim_hr@yahoo.co.id

  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].

  Previous research on ethanol production from water hyacinth revealed that it can be successfully converted to ethanol without much difficulty. Such research includes methods and conditions of pretreatments; types, ratio and concentration

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  AN CMC Carboxymethyl Cellulose CU DAP Dilute Acid Pretreatment DNS Dinitrosalicylic acid FPU Filter Paper Unit GB LHW Liquid Hot Water PDA SC TR 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

  o

  container at 8 °C, were grown in potato dextrose agar (PDA) at 20

  C. Prior to

  o

  usage, SC was warmed to 20 C for 30 min, whilst TR, AN, and CU

  o

  were inoculated at 20 C for 2 days (1 day for CU) in liquid media containing 22% sucrose, 1% KH

  2 PO 4 , and 1% (NH

4 )

2 SO 4 [26]. All procedures were done aseptically.

  ' ! "

  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].

  $ %

  The hydrolysis and fermentation were carried out simultaneously. After pretreatment, 30 ml enzymes, 001.23 g (000.1 M) MgSO , and 0.5 g (1% w/v) SC

  4

  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

  4 was kept in range of 00100 mM to observe the

  effects on fermentation at higher concentration as studies on this subject suggest that MgSO has positive effect at concentration of 3.5 mM but becomes inhibitory

  4

  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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  Water hyacinth was analysed for its moisture content and major constituents (hemicellulose, cellulose, and lignin). Moisture content analysis was carried out by gravimetric method. Hemicellulose, cellulose, and lignin content were

  '

  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].

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  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 against calibration curve. Iso0propanol was used

  4

  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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  37.50 34.19 32.69 ± 0.024 22043.4

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  27.78 17.66 19.02 ± 0.017 17.8031

  5

  5.99 12.22 4.37 0.027 7026.36

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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.

  Out of all pretreatments performed in this study, DAP yielded the highest amount of sugar and ethanol. For DAP, a substantial amount of sugar was not converted. This is because hydrolysis of hemicellulose yields pentose which cannot be utilized by SC. In both simple sterilization and LHW, the results were comparable in terms of ethanol and sugar yield. It might be due to the close operational condition. For biological pretreatment, despite higher sugar release, ethanol concentration was lower. While higher sugar release might be related to additional enzymes from white rot fungus (GB), lower ethanol concentration might be caused by inhibition of SC by GB, or conversion of either ethanol or sugar to other products. The latter case was more probable as GC analysis on biologically pretreated broth sample indicated existence of an unknown volatile compound in form of third peak.

2.0 Sterilization DAP LHW Biological

  85 / + Sugar Ethanol

  85 / - / Sugar Ethanol

  

50

75 100 ' -

  25

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  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.

  Magnesium sulfate increases yeast tolerance on alcohol by decreasing cell membrane permeability [27]. Salts in general also induce salting out effect on water

  7 !

• – 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.

  '

  A higher MgSO

  4 concentration could not be tested because the salt disturbs 2+

  sugar analysis by DNS method as shown in Fig. 3. The existence of Mg 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].

  6

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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.

  1.2 Sugar, 25mM Sugar, 50 mM /

  Ethanol, 25 mM

  0.9

85 Ethanol, 50 mM -

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  3

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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].

  '# # !

  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.

  3 Sugar, 25 mM Ethanol, 25 mM /

  Sugar, 50 mM

  85 -

2 Ethanol, 25 mM

  1 '

  A B C 6 # ! ' ! ; ' : <; ' 6 '= : '; ' > ' '= 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.

   '

  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.

  '6

  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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