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Comparative of Biomass for PretreatmentWith Biological Process for

Efficient Hydrolysis

Desy Kurniawatia, Muhammad Natsira, Rahmi Febrialisa, Prima Endang Susilowatia

Abstract

The production of ethanol from sugar and starch is a well-known technology, while making ethanol from lignocellulosic materials like grass, sago dregs and sawdust, is more difficult and requires more process steps. Many physicochemical structural and compositional factors hinder the hydrolysis of cellulose present in grass, sago dregs and sawdust to sugars and other organic compounds that can later be converted to fuels. Biological pretreatment techniques change the physical and chemical structure of the lignocellulosic biomass and improve hydrolysis rates. Methods involving the disruption of enzyme and plant cell wall polysaccharide interactions were developed to recover xylanase, cellulase, and endoglucanase activity from deconstructed solids. The present study was aimed to hydrolysis grass bio wastes, sago dregs and sawdust, using microbial from Wanggu River. The result show that the optimum conditions of the fermentation obtained 1% with 48 hours incubation time. Analysis of reducing sugar for both concentration and optimum time were 91 mg/mL (sago dregs), 121 mg/mL (grass), and 108 mg/mL (sawdust).

Keywords: Fermentation, Grass, Microbial, Sugars

aChemistry Department, FMIPA, Halu Oleo University, Kendari,Indonesia

Corresponding author email address: echimopifree@yahoo.co.id

Introduction

Availability of lignocellulosic materials in Indonesia is quite a lot, including in the Southeast Sulawesi, this is in line with the development of agriculture and forestry industries that produce lignocellulosic wastes. for example, the lignocellulosic waste sago pulp, reeds and sawdust that is effluent from corn starch processing industry, furniture industry and agricultural weeds.

The presence of grass, sago waste and sawdustin Southeast Sulawesi is very abundant and sometimesintrusive. This biomass growth is very fast. During this waste not fully utilized. Grass, sago waste and sawdust contains lignocellulose. The main components of lignocellulose is lignin, cellulose, and hemicellulose [1]. Bioethanol is ethanol derived from all types of biomass containing sugar, starch and lignocellulose that has potential as a replacement for conventional fuel oil [2]. Raw materials that have been developed for the production of bioethanol for is sugar cane, sugar beet, tapioca, sorghum, meizena, barley, wheat, rice and potatoes. But the source of biofuels to compete with its use as food and feed. Therefore, we need to utilize lignocellulosic waste as a source of non-food BBM for producing bioethanol fuel.

Energy has an important role and cannot be separated in human life. Utilization of non-renewable energy in excess lead to an energy crisis. One of the symptoms

of the energy crisis is scarcity of fuel oil (BBM). One alternative to overcome the scarcity of energy, is by the pattern of waste management biomass, so that the biomass can be utilized to process into ethanol. Bioethanol is produced can be used as an energy source, whereas a byproduct of organic fertilizers can be used for crops. So expect environmental issues and energy shortages can be resolved.

This research aims to compare of biomass for pretreatmentwith biological process for efficient hydrolysis so that can behydrolysed into monosaccharides, namely xylose and glucose.Forfermentation of monosaccharides (xylose and glucose mixture) into bioethanol done with the help of yeast.

Methodology

A. Grass

Grass is a kind of nuisance weeds and cultivated plants as a major source of harm to fire on cultivated plants and forests. The presence of grass that is considered harmful and disturbing this was not what people expected so far. During this time the grass has been used as fodder, roofing houses, paper mills materials, craft materials. Grass is a lignocellulosic material that is mostly composed of cellulose, hemicellulose, and lignin (Table 1).

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B.Sago Waste

Sago waste is a waste by product of processing sago starch. Comparison of the amount of starch and sago residues in corn starch processing is 1: 6. The sago waste that much has yet to be exploited. Sago waste causing pollution and sago bark is mostly lignocellulosic materials composed by cellulose, hemicellulose and lignin.

Table 1. Chemical Composition of Grass [3]

Composition of Grass Presentation (%)

Water 93.76

Extractive 8.09

Lignin 311.29

Alpha cellulose 40.22

Pentose Hemicellulose 181.40

Table 2.Sago Waste Composition[4]

Component Sago waste(%)

Cellulose 19.55

Residue lignin 20.67

Pentose sugar 11.70

Extractive 10.60

Ash 6.89

C.Sawdust

Sawdust is a wood waste from sawmills. The more wood industry (furniture), the more waste is generated (sawdust). During this sawdust is only allowed to accumulate. Sawdust still contains a lot of cellulose that can be harnessed into useful products, such as for the production of biogas. The composition of sawdust from hardwoods wood types shown in Table 3.

Table 3.Sawdust composition [5]

Component % weight

Lignin 27

Cellulose 42

Hemicellulose 18

D.Lignocellulose

Lignocellulose is composed of carbohydrate polymers, namely cellulose, hemicellulose, lignin and little parts (salt and minerals). Cellulosic is forming frame of biomass of woodsurrounded by hemicellulose and lignin (Figure 1). Hemicellulose and lignin matrix serves as a lining material. Cellulose and hemicellulose are polysaccharides that can be hydrolysed into sugars [6].

Cellulose is composed of units homopolysaccharide-D-glucopyranose are bonded to each other withbound β-1, 4 glycoside (Figure 2) [6]. According Achmadi [7], cellulose has two kinds of hydrogen bonds, the intermolecular hydrogen bonding and intermolecular hydrogen bonds. Intra-molecular hydrogen bonds that maintain the strength of the cellulose chain and intermolecular bonding which causes the cellulose chains bind to each other to form a fibril micro fibrils. Structure and strong hydrogen bonds, leading to cellulose is not soluble in various solvents.

Figure 1. Schemes cell wall of woody plants

Figure 2.Molecular Structure of Cellulose

Hemicellulose is a complex polymer consisting of a mixture of polymers of monosaccharides. Grouped based on their constituent sugars are hexoses (glucose, mannose and galactose), pentoses (xylose, arabinopiranosa, arabinofuranosa), hexuronic acid (glucoronic, methylglucuronic, galactoronic), and deoxy hexoses (ramnose, fructose) [8]. Therefore, hemicellulose is defined as short-chain heteropolysaccharide consisting of a mixture of hexoses and pentoses.

The largest component of hemicellulose is xiloglucan, which composed of glucose units with bound β-1-4 which ends form a branch of the xylose units on the bond α-1-6. This molecule is covalently bound to the cell wall pectin fraction and hydrogen bonds to the cellulose microfibrils so as to strengthen the plant cells.

E. Bioethanol

Bio-ethanol (C2H5OH) is the liquid from the fermentation of sugars from carbohydrate sources using the help of microorganisms. Bioethanol is produced from starch food by fermentation.

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Bioethanol is a fuel from vegetable oils that have properties resembling premium oil [1].

Reactions that occur in the bioethanol production process are simple as follows:

F. Optimization of Hydrolysis Conditions delignification and hemicellulose

Wanggu bacterial isolates were grown in media containing various concentrations of the substrate with the grass. Fermentation is done at room temperature with incubation time was varied. Results were then analysed degree lignin degradation, decrease in dry weight, levels of cellulose, hemicellulose content and reducing sugar levels.

Determination of optimum substrate types performed by growing the bacteria in 1 mL in 20 mL of LB medium containing 2% lignocellulosic waste (sago pulp, sawdust and reeds). Next cultures were incubated at room temperature for 48 hours. Medium was filtered with filter paper, then the precipitate was rinsed with 40 mL H2O 3 times treatment, the residue was dried in an oven at a temperature of 105 °C to constant weight prior to the analysis of the degree of degradation of lignin.

Determining the optimum hydrolysis time for potential bacterial production delignification performed by growing the bacteria in 1 mL in 20 mL of LB medium containing lignin from lignocellulosic wastes. Subsequently cultures were incubated at room temperature for 6-36 hours. Media was filtered with filter paper, then the precipitate was rinsed with 40 mL H2O 3 times, the residue was dried in an oven at a temperature of 105 °C to constant weight prior to the analysis of the degree of degradation of lignin.

Determination of Optimum SubstrateConcentration is done by each of the selected bacteria were grown in liquid LB medium and then transferred in liquid media containing lignocellulosic substrates with concentrations of 0%, 1%, 5% and 10% (sago pulp, reeds and sawdust). Furthermore inoculated at room temperature for 1 and 2 days. Media was filtered with filter paper. Furthermore, the precipitate was rinsed with 40 mL H2O 3 times, the residue is dried in the air freely.

The degree of Lignin Degradation Analysis was done by weighing 0.5 g of dry residue incorporated into erlenmeyer 100 mL, was added 10 ml of 0.02 M KMnO4 and 2.5 ml of 2.0 M H2SO4, and then in the shaker at a

speed of 200 rpm for 15 minutes. The solution was filtered and the filtrate was determined by spectrophotometer absorbance at a wavelength of 546 nm.

G. Analysis of Waste Lignocellulose After delignification

The residue is dried and weighed in analytical balance. Furthermore, the determination of the percentage loss of substrate dry weight can be calculated based on the formula:

Substrateweight reduction% =𝐵𝐾𝑂 𝑑𝑎𝑦 0−𝐵𝐾𝑂 𝑑𝑎𝑦 𝑡

𝐵𝐾𝑂 𝑑𝑎𝑦 0𝑥 100

Description:BKO=oven dryweight(g) t=time ofincubation(1 and2days)

Determination ofthe rate ofdecompositionof lignocellulosicwastescan be calculatedbythe formulaas follows:

𝑘 =𝑙𝑛

𝑋𝑡

𝑋0

𝑡

Description: k=decomposition rate ofsubstrate

Xt=Ovendry weight-0 (g)

X0=dry weighttooven-t (g)t=1and2days

t=lengthof incubationtime

Levels holocellulose made by TAPPI T 9 m-54 and determination of cellulose made by TAPPI T 17 m-55 [6]. Levels obtained by reducing the hemicellulose content of the cellulose content holocelluloce. Determination of reducing sugar made by the DNS method [6].

Results and Discussion

Percentage decrease in dry weight of biomass is one measure of the effect of biodegradation by bacteria lignolytic. Lose dry weight fast because of lignocellulosic wastes in bacterial growth media it will be hydrolysed by bacteria. Results hydrolysis of lignocellulosic wastes can be used to obtain energy and constituent bacterial cells. This is what causes the weight loss of lignocellulosic wastes in growth media. Incubation treatment isolates W.1.3 on grass has led to decrease in dry weight (Figure 3)

Degradation rate associated with a decrease in the rate of decomposition of the dry weight per unit time. The rate of decomposition of the biomass, during the time of incubation of bacteria also increased compared to the control (without the addition of bacteria) (Figure 4).

Research data shows the level of degradation and decomposition rates were highest after 48 hours of incubation. It shows the rate of growth of bacterial

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cells are increased in the process of lignin degradation in lignocellulose waste media and increased produce of enzymes

Figure 3. Rate of degradation of lignocellulosic waste. Description: (a) 24-hours incubation, and (b) 48 hours incubation

Figure 4. Waste Decomposition rate of lignocellulose. Description: (a) 24-hour incubation, and (b) 48 hours incubation

In addition to the length of the incubation period, the level of degradation and decomposition rate is influenced by the concentration of waste lignocellulosic substrate will be degraded by bacteria. Results showed that the concentration of 1% gives better results than at concentrations of 5% and 10% (Figure 3 and 4). It means that the fewer the number of lignocellulosic waste, bacteria would be easy to degradation of lignocellulosic. It can be concluded the rate of increase in the rate of degradation of lignocellulosic waste decomposition by bacteria lignolytic influenced by the long incubation time and substrateconcentration.

A. Holocelluose

The test is performed to determine the percentage of fiber content section free of lignin, which is composed of cellulose and hemicellulose. Figure 5 showed levels holocellulose of biomass after incubation for 24h and 48 h.

During the incubation period, levels holocellulose of sago waste 1.5%, Grass 24.57% and Sawdust 23.11%range. This increase occurred because of reduced levels of lignin in the biomass, thus increasing the number holocellulose. Reduced levels of lignin in biomass occurs because bacterial activity lignolytic issued by ligninase enzymes that can degrade lignin. Holocellulose levels greatly affect the incubation time and substrateconcentration to a given onlignolytic bacterial. Incubation time was good for bacteria to degrade lignin is 48 hours with a concentration of 1%.

B. Cellulose

Biomass cellulose content ranged from 2.8% (sago waste), 19.94% (Grass) and 21.22% (sawdust) increase. Highest levels increased cellulose obtained after 48 hours of incubation with 1% concentration. Increased levels of cellulose occurs because of lack of levels of lignin in lignocellulosic wastes, thus increasing free cellulose.

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Figure 5. Holocellulose levels of lignocellulosic wastes. Description: (a) 24-hour incubation, and (b) Incubation 48 hours

Figure 6.Cellulose levels lignocellulosic waste. Description: (a) 24-hour incubation, and (b) Incubation 48 hours

C. Hemicellulose

During the incubation period, hemicellulose levels also rise 0.01% (Sago waste) , 4.62% (Grass) and 1.94% (Sawdust) range. Highest levels hemicellulose obtained after 48 hours of incubation with 1% concentration. Increased levels of hemicellulose occurs because of lack of levels of lignin in lignocellulosic waste. This data is somewhat different from the results of the study reported by Aisah [6] levels holocellulose, cellulose and hemicellulose increased with incubation time of 6 days to degrade lignin by fungi P. chrysosporium.

Results obtained showed that the bacterial isolates wanggu river has the ability to degrade lignin and break the bonds between lignin cellulose and hemicellulose.

D. Analysis of reducing sugar

This hydrolysis produces the effect of the level of glucose in the hydrolysate grass. Figure 8 shows that the resulting reducing sugar from hydrolysate of Biomass, most of the incubating bacteria at 48 hours with substrate concentration of 1%.

Figure 7.Hemicellulose levels lignocellulosic wastes. Description: (a) 24-hour incubation, and (b) Incubation 48 hours

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Figure 8. Levels of reducing sugar Against Incubation time with Bacteria Treatment at Different concentrations of substrate Description: (a) 24-hour incubation, and (b) Incubation 48 hours.

Based on data showing that reducing sugar lignocellulosic wastes generated relatively high (Figure 8) therefore the obtained hydrolysate can be fermented to produce ethanol. Concentration and time delignification of lignocellulosic waste the most excellent of the data obtained at a concentration of 1% by 48 hours. Analysis of reducing sugar at the optimum time and concentration of 121 mg/mL. Reducing sugar produced in this study is much larger than the research of Devis (2008) [6]which produces reducing sugar by 16 mg/mL.Hartoto [4]stated that the concentration of sugar is good for ethanol fermentation is 16 to 25%, which will produce ethanol by 6 to 12% (w/ v)

Conclusions

Data have been obtained conditions on delignification and hemicellulose hydrolysis Biomass. Where the concentration and time delignification of lignocellulosic waste the best is the concentration of 1% by 48 hours. Analysis of reducing sugar of biomass at the optimum time and concentration of 91 mg/mL (Sago waste), 121 mg/mL (Grass) and 108 mg/mL(Sawdust).

Acknowledgments

This work was supported by Program Hibah Bersaing BOPTN Dirjen Pendidikan Tinggi of Indonesia, Halu Oleo University. The authors would like to thankChemistryOn International Conference 2014, Students of Biochemistry On Chemistry Department FMIPA UHO for helpful discussions, Biochemistry Laboratory FMIPA UHO.

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