Pretreatment of garden biomass by alkali-assisted ultrasonication: effects on enzymatic hydrolysis and ultrastructural changes
© Gabhane et al.; licensee BioMed Central Ltd. 2014
Received: 9 May 2013
Accepted: 12 April 2014
Published: 28 April 2014
The present investigation aims at studying the effectiveness of alkali-assisted ultrasonication on pretreatment of garden biomass (GB). Dry and powdered GB suspended in 1% NaOH was ultrasonicated for 15, 30 and 60 minutes at a frequency of 25 KHZ. The mode of action and effectiveness of alkali-assisted ultrasonication on GB was established through microscopic, scanning electron microscopic and X-ray diffraction studies. A perusal of results showed that alkali-assisted ultrasonication led to fibrillation of GB which ultimately facilitated enzymatic hydrolysis. The results also indicated that alkali-assisted ultrasonication is an efficient means of pretreatment of GB at moderate (45-50°C) working temperature and low (1%) concentration of alkali. The yield of reducing sugar after enzymatic hydrolysis increased almost six times as compared to control due to alkali-assisted ultrasonication.
KeywordsUltrasonication Alkali pretreatment Garden biomass Enzymatic hydrolysis Cellulosic ethanol Ultra structural changes
Lignocellulosic substrates are potential sources for the production of ethanol through microbial intervention because they are abundant, cheap and renewable . The process of conversion of lignocelluloses into glucose is through hydrolysis for which the lignin bound to xylan and glucomannan [2, 3] is known to be a recalcitrant compound. Pretreatment is therefore necessary to delignify and facilitate the disruption of lignocellulosic moiety. Pretreatment alters the structure of cellulose and making it more accessible to the enzyme that convert carbohydrate polymer into fermentable sugar [4, 5]. Thus, the general idea of pretreatment is to increase cellulose accessibility, which can be done by removing or altering hemicelluloses or lignin, decreasing the crystallinity of cellulose and increasing the surface area. Until now, the overall conversion of cellulose material into glucose has been hampered mostly by economic problems such as high cost of pretreatment. Hence, cost effective but efficient means of lignocellulosic pretreatment is crucial for viable production of cellulosic ethanol.
Generally, pretreatment methods are either physical or chemical. Some methods incorporate both effects . Most of the conventional pretreatment processes utilize heat (thermal energy) to mediate bond breaking between molecules during chemical action. Different kinds of heating devices such as autoclave, microwave digesters, heating coils etc. are used in pretreatment processes. The energy consumption of these heating devices is normally high that ultimately affects cost effectiveness of cellulosic ethanol production. Dilute acid pretreatment has been widely studied because it is effective and relatively inexpensive . Steam pretreatment (with pressure) is also one of the preferred methods of hydrolysis of lignocellulosic feed stocks. Steam pretreatment supplies moist heat under pressure that results in substantial breakdown of lignocellulosic structure, hydrolysis of hemicellulosic fraction, depolymerisation of lignin components and defibration . Microwave irradiation has also been employed for lignocellulosic pretreatment [9, 10]. Microwave generates thermal energy through dielectric heating  that alters the ultra structure of cellulose, degrade lignin and hemicellulose and increase the enzymatic susceptibility of reducing sugar .
Ultrasonication has seen wide application in cell crushing, removal of dead cell in surgery  and breaking filamentous algae in small pieces . Sonication is the act of applying ultrasound energy to agitate the particles and to speed up dissolution of molecules by breaking the intermolecular interaction. Ultrasonication can be a promising alternative to conventional hydrolysis methods . The ability of ultrasonication in degrading polymeric sequences has been well documented, particularly in synthetic materials dissolved in various solvents  and in extracting lignin and hemicellulose from lignocellulosic materials [17, 18]. Some studies have suggested that pretreatment of lignocellulose substrates with ultrasonication could be useful for the intensification of bioconversion both in nature and under production condition .
Although reports [20–23] are available relating to the effects of ultrasonication on lignocellulosic disruption, there is paucity of information about the effectiveness of alkali-assisted ultrasonication, particularly on garden biomass. Garden biomass (GB), a potential cellulosic resource for bio energy production is commonly found in urban waste. It is rich in recalcitrant molecules such as cellulose and lignin, and relatively small amounts of saccharides, amino acids, and proteins. As GB is rich in cellulose, it can be used as a raw material for bio energy production after pretreatment.
Therefore, in the present study, we aimed to assess the efficacy of alkali-assisted ultrasonication on the pretreatment of GB. The major objectives of present investigation are: i) to study the effectiveness of alkali-assisted ultrasonication on ultra structure and delignification of GB ii) to assess the effectiveness of alkali-assisted ultrasonication on enzymatic hydrolysis and ethanol production.
Material and methods
Collection and processing of garden biomass
Garden biomass (GB) consisting mostly grasses (85-90%) of the species of Cynodon ductylon and Elusine indicus and small portion of weeds and fallen leaves was collected from the garden area of National Environmental Engineering Research Institute (NEERI) and sun dried for 2–3 days followed by oven drying at 70°C for about 92 hours. The dry GB was powdered using a pulverizer to pass a 1 mm sieve and kept in polyethylene sample containers inside a wooden cupboard at room temperature (28 ± 2°C) for further experiments. Known quantity of this powdered material was analyzed for initial composition and the remaining used for further experiments.
Alkali –microwave pretreatment
Two gms of dry and powdered GB was taken in a 500 ml conical flask and suspended with 100 ml of 1% NaOH (W/V). The contents were mixed well and digested in a microwave digester (Ethos 900, Italy) at 180°C, 700 W for 30 minutes. The digested material was cooled and subjected for compositional analysis as per section 2.4.
Two gms of dry and powdered GB was taken in a 500 ml conical flask and was suspended with 100 ml of NaOH in three different (0.5, 1.0, and 5.0% ) concentrations (W/V). The contents were mixed well and sonicated for 15,30 and 60 minutes at 25 KHZ frequency with an effective ultrasonic power of 150 W using an ultrasonicator (Lark, USA). The control treatment received no sonication. The resultant liquid after ultrasonication was analyzed for reducing sugar, which was totaled, with the reducing sugar of enzymatic hydrolysis later on. The residue after repeated washings with de-ionized water was dried in an oven at 50°C for 48 hrs and analyzed for different parameters as per section 2.4.
Compositional analysis of GB before and after pretreatment
Samples of GB (dry and powdered) collected before and after pretreatment were analyzed for cellulose content using its hydrolyzed residues by HNO3-ethanol method . The hemicellulose content was analyzed by Liu method . The lignin content of GB was estimated according to H2SO4 method . The yield of reducing sugar after enzymatic hydrolysis was estimated by DNS method  using glucose as standard.
Polarized light microscopic (PLM) and Scanning electron microscopic (SEM) studies on pretreated GB
The impact of alkali-assisted ultrasonication on surface morphology and ultra structure of both alkali-microwave and alkali-assisted ultrasonication pretreatment of GB was studied using polarized light microscope (Olympus, BX-80) and scanning electron microscope (JEOL-JED, Japan), respectively. The pretreated GB samples were dispersed in distilled water. The suspension was dropped and mounted on a glass slide and viewed through PLM. For scanning electron microscopic studies, samples of GB after pretreatment were mounted on glass slides, dried at 45°C in an oven before fixing it on stubs. The stubs were then coated with platinum by an ion sputter and imaged through SEM.
XRD and crystallinity measurements
Where Cr I is the crystallity index, Icr is maximum diffraction intensity at peak position 2^ ~ 22.6° and Iam is the intensity at 2^ = 18.7°
The residue of GB after pretreatment was washed thoroughly with de-ionized water and enzymatically hydrolyzed using a commercial cellulase (pure) enzyme, ONOZUKAR-10 procured from Hi-Media, India. The solid loading rate for enzymatic hydrolysis was 6 g/100 ml (0.05 M citrate buffer) with an enzyme loading rate of 50 mg (50 FPU)/g of GB. The pH during enzymatic hydrolysis was 4.8 and the temperature maintained at 50°C using a shaker incubator operated at 150 rpm for 48 hrs. 2.5 mg of Tetracycline was also added to avoid microbial contamination during enzymatic hydrolysis.
All experiments were replicated thrice and mean values with standard deviation presented in tables. The characterization of GB before and after pretreatments were statistically analyzed using one way ANOVA and Duncan’s Multiple Range Test (DMRT) using SPSS software (version 11.5)
Results and discussion
Characterization of garden biomass (GB)
Composition of garden biomass expressed as % of dry matter
Total organic matter
Effectiveness of alkali concentration and reaction (ultrasonication) time on enzymatic hydrolysis of GB
Effectiveness of alkali concentration and sonication time on reducing sugar yield after enzymatic hydrolysis
Time (min) and concentration (%) of alkali
Reducing sugar yield after enzymatic hydrolysis
% increase/decrease in reducing sugar concentration
5.40 ± 0.65 a
26.53 ± 0.76 c
32.76 ± 1.92 d
39.26 ± 0.78 ef
7.64 ± 2.33 ab
31.05 ± 3.34 d
37.71 ± 1.23 e
41.63 ± 0.62 f
8.41 ± 1.49 ab
36.54 ± 1.94 e
48.13 ± 1.57 g
52.32 ± 0.80 h
9.55 ± 1.58 b
42.42 ± 1.68 f
59.56 ± 2.60 j
56.40 ± 3.43 i
The increase in reducing sugar yield was significantly high only when ultrasonication was assisted by alkali. On the other hand, pretreatment of GB with either with alkali or ultrasonication alone did not cause any increase in reducing sugar yield. Ultrasonication of GB for 60 minutes without alkali yielded 9.55% of reducing sugar after enzymatic hydrolysis, whereas ultrasonication with alkali (1%) yielded 59.56% of reducing sugar, more than six fold increase in reducing sugar over ultrasonication alone treatment. Similarly, 1% alkali alone yielded 32.76% of reducing sugar which is much lesser than the yield (59.56) of alkali assisted ultrasonication.
Initial and final characterization of garden biomass after alkali- microwave and alkali-sonication pretreatments
Initial concentration (%)
Final concentration (%)
Alkali + Microwave
Alkali + Sonication
44.03 ± 1.39 a
66.25 ± 2.83 b
73.08 ± 0.77 c
77.28 ± 0.85 d
28.55 ± 3.19 c
12.85 ± 0.69 b
7.19 ± 0.32 a
8.02 ± 0.85 a
24.46 ± 0.64 d
19.80 ± 0.82 c
15.86 ± 0.87 b
13.39 ± 0.69 a
Effects of alkali-assisted ultrasonication on ultra structural changes of garden biomass
Effect of alkali-assisted ultrasonication on crystallinity of cellulose in garden biomass
The measurement of crystallinity index is important because ultrasonic treatment of cellulose generally cause a strong decrease in the degree of polymerization . A perusal of results showed an increase (0.828) in CrI of alkali-ultrasonicated GB over the control (0.729). However, comparing to alkali-microwave (0.848) pretreatment, this is slightly low, might be because of high temperature (180°C) involved in alkali-microwave pretreatment that effectively converted maximum of amorphous fraction of cellulose into reducing sugars and exposing the crystalline fraction more prominently.
Based on results, it may be concluded that alkali-assisted ultrasonication is one of the effective methods of pretreatment for GB as it increases the net yield of reducing sugar after enzymatic hydrolysis. The microscopic studies further revealed that alkali-assisted ultrasonication caused fibrillation of cellulosic moiety, which ultimately favored enzymatic hydrolysis process. Alkali-ultrasonication was carried out at moderate temperature, at which further degradation of reducing sugar is not possible, an advantageous condition for alcohol fermentation.
Solid and Hazardous Waste Management Division, National Environmental Engineering Research Institute, Nehru marg, Nagpur, Maharashtra, INDIA.
Filter paper unit
Polarized light microscope
Scanning electron microscope
The work is a part of the outcome of CSIR- Supra Institutional Projects in Molecular Environmental Sciences (SIP-MES, Activity 4.8). We thank Dr. Saravana Devi, Dr. Sadhana Rayalu and Dr. Nitin Labhsetwar for helping us in microscopic and XRD studies. We also thank Dr. Nitin Dongarwar for helping us in the identification of grass species used in the present investigation.
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