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Sequential low and medium frequency ultrasound assists biodegradation of wheat chaff by white rot fungal enzymes
Accepted Manuscript Title: Sequential low and medium frequency ultrasound assists biodegradation of wheat chaff by white rot fungal enzymes Author: Christine M. Oliver Raymond Mawson Laurence D. Melton Geoff Dumsday Jessica Welch Peerasak Sanguansri Tanoj K. Singh Mary Ann Augustin PII: DOI: Reference:
S0144-8617(14)00385-3 http://dx.doi.org/doi:10.1016/j.carbpol.2014.04.028 CARP 8789
To appear in: Received date: Revised date: Accepted date:
27-9-2013 31-3-2014 8-4-2014
Please cite this article as: Oliver, C. M., Mawson, R., Melton, L. D., Dumsday, G., Welch, J., Singh, P. S., Tanoj K., & Augustin, M. A.,Sequential low and medium frequency ultrasound assists biodegradation of wheat chaff by white rot fungal enzymes, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.04.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sequential low and medium frequency ultrasound assists
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biodegradation of wheat chaff by white rot fungal enzymes
Christine M. Olivera, Raymond Mawsona, Laurence D. Meltonb, Geoff Dumsdayc,
CSIRO Animal, Food and Health Sciences, 671 Sneydes Road, Werribee, VIC 3030,
us
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Jessica Welchc, Peerasak Sanguansria Tanoj K. Singha, Mary Ann Augustina*
Australia
Food Science, School of Chemical Sciences, University of Auckland, Private Bag
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b
92019, Auckland, New Zealand c
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CSIRO Material Science and Engineering, Normanby Road, Clayton, VIC 3168,
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Australia
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*Corresponding author. Address: CSIRO Animal, Food and Health Sciences, 671
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Sneydes Rd, Werribee, VIC 3030, Australia. Tel.: +61 3 9731 3486; fax +61 3 9731 3201.
E-mail address: [email protected]
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Abstract The consequences of a short (10 min per frequency) ultrasonic pre-
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treatment using low (40 kHz), medium (270 kHz), or sequential low and medium
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frequency (40 kHz followed by 270 kHz), on the degradation of wheat chaff (8 g 100
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ml-1 acetate buffer, pH 5) were evaluated. In addition, the effects of the ultrasonic
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treatment on the degradation of the wheat chaff when subsequently exposed to
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enzyme extracts from two white rot fungi (Phanerochaete chrysosporium and
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Trametes sp.) were investigated. Pre-treatment by sequential low and medium
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frequency ultrasound had a disruptive effect on the lignocellulosic matrix. Analysis of
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the phenolic-derived volatiles after enzymatic hydrolysis showed that biodegradation
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with the enzyme extract obtained from Phanerochaete chrysosporium was more
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pronounced compared to that of the Trametes sp. The efficacy of the ultrasonic pre-
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treatment was attributed to increased enzyme accessibility of the cellulose fibrils due
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to sonication-induced disruption of the plant surface structure, as shown by changes in
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the microstructure.
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Keywords Ultrasound, White rot fungal enzymes, Phanerochaete chrysosporium,
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Trametes sp., Wheat chaff, Biodegradation
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1.
Introduction
20 Lignocellulosic biomass is an abundant and renewable source of fuel, energy and organic chemicals and has potential application as an animal feed (Sun, 2010). Pre-
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treatment of lignocellulosic biomass is necessary to alter its physical and chemical
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structure for improved cellulose/hemicellulose accessibility (Weng, Li, Bonawitz, &
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Chapple, 2008; Zakzeski, Bruijnincx, Jongerius, & Weckhuysen, 2010; Volynets &
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Dahman, 2011). Current pre-treatment methods used to degrade lignocellulosic
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biomass involve the utilization of harsh chemicals (e.g. strong bases, concentrated
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sulphuric acid, oxidizing agents) (da Costa Sousa, Chundawat, Balan, & Dale, 2009)
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or energy-intensive technologies such as steam explosion (Josefsson, Lennholm, &
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Gellerstedt, 2001; Hana, Deng, Zhang, Bicho, & Wue, 2010). Biological pre-
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treatment of lignocellulosic biomass is an alternative more environmentally-friendly
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approach (Leonowicz et al., 1999; Sánchez, 2009). The most promising
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microorganisms for biological pre-treatment are basidomycetes, which degrade lignin
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to varying extents depending on the type of fungi and fermentation conditions used (Dinis et al., 2009). These fungi secrete one or more of three extracellular enzyme
complexes that are fundamentally important for lignin degradation, predominantly lignin peroxidase, manganese peroxidase, and laccase (Hatakka, 1994; Jönsson &
Nyman, 1992). Even so, biodegradation of lignin by white rot fungi alone is itself an
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impractically slow process. Alternative pre-treatment approaches, which not only
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reduce the energy input and the use of harsh chemicals but increase the total process
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efficiency of biomass transformation, are required.
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Microwave irradiation has been investigated as an alternative means of pretreating lignocellulosic material. For example, Binod et al. (2012) showed that
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microwave-based alkali pre-treatment of sugarcane baggase removed external fibres,
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increasing the accessibility of the cellulose to enzymes. Chen, Tu & Sheen (2011)
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found that dilute acid pre-treatment of ground sugarcane bagasse using microwave
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heating markedly destroyed the lignocellulose structure, causing swelling and
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fragmentation of the bagasse particles. The ability of microwave to facilitate the
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extraction of soluble phenolic compounds from plant material has also been reviewed
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(Routray, & Orsat, 2012).
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Ultrasound has been used successfully in conventional processes of desizing,
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extraction and wool wax scouring (Yachmenev, Bertoniere, & Blanchard, 2001;
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Yachmenev, Blanchard, & Lambert, 2004; Hurren, Zhang, Liu, & Wang, 2006).
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Typically, in such applications, mechanically pre-comminuted material is placed in a
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medium containing either strong acid or alkali and subjected to a single low frequency
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over the range 20−40 kHz. Sun & Tomkinson (2002) demonstrated ultrasound (20
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kHz) assisted alkali-dissolution of ground wheat straw. Similarly Velmurugan &
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Muthukumar (2001) applied ultrasonication (24 kHz) to assist alkali delignification of
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powdered sugarcane bagasse by increasing surface oxidation of lignin and
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hemicellulose. Li, Sun, & Sun (2010) showed that ultrasound treatment (35 kHz) of ball-milled, dewaxed bamboo culms increased the partitioning of lignin into ethanol-
soluble extracts. Pre-treatment of sawdust with ultrasound (22 kHz) enhanced lignin
degradation during subsequent solid-phase cultivation of Panus (Lenitnus) tigrinus (Kadimaliev, Revin, Atykyan, & Samuilov, 2003). It has also been established that
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ultrasound in combination with appropriate solvents can be used to recover lignin
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from hemicelluloses (García, Alriols, Llano-Ponte, & Labidi, 2011).
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Ultrasound also has the potential to increase the activities of enzymatic processes.
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For example, ultrasound (16 and 20 kHz, up to 3 W cm-2) has been shown to enhance
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the enzymatic bio-scouring of cotton (Yachmenev et al., 2004). While the disruptive
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effects of cavitation from low frequency ultrasound (20−100 kHz) physically tease the
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plant material apart and increase accessibility to the underlying cellulose, higher
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frequencies (>100 kHz) increase free radical generation and therefore may be
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expected to sonochemically oxidize components (Ashokkumar et al., 2008).
The primary objective of this work was to evaluate the impact of low and medium
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frequency ultrasound on wheat chaff and its subsequent biodegradation by enzyme
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extracts obtained from cultivation of two white rot fungi, Trametes sp. and
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Phanerochaete chrysosporium. It was hypothesized that the use of a combination of
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frequencies would be more effective than a single frequency for disrupting plant
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biomass, thereby improving enzyme access to the underlying cellulose. The
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justification for using 40 kHz as the initial pre-treatment frequency in the sequential
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process presented here was based on the scouring of wool wax, where 35–45 kHz was
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found to be optimal for cracking the waxy layer and stripping away the scaly surface
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(Hurren et al., 2006). Additionally targeting of the cracks in the waxy cuticle and
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potential lifting of the waxy surface by micro-streaming could be expected. The
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second frequency of 270 kHz was selected to provide enhanced oxidizing potential (Ashokkumar et al., 2008) and specifically erode the closed lip crevice of the microscopic respiratory stomata, and thereby enhance accessibility of enzymes to the internal tissue structure. Based on previous literature findings using microwave (Chen
et al., 2011; Binod et al., 2012), a brief high temperature heat treatment of the wheat
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chaff prior to ultrasonic pre-treatment was also evaluated for its ability to enhance
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sonication-induced disruption of the lignocellulosic material and subsequent
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enzymatic hydrolysis.
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2. Materials and Methods
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2.1 Growth of the fungal isolates and preparation of the enzyme extracts
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The Trametes sp. strain 2403 and Phanerochaete chrysosporium strain 16543
were obtained from the CSIRO Collection of Wood Decaying Fungi. The medium
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used to grow the fungal isolates contained (per litre): 3 g NaNO3; 1 g K2HPO4; 0.5 g
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KCl; 0.5 g yeast extract (BD Bacto) and 0.01 g FeSO4.7H2O. After formulation the
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medium was adjusted to pH 5 using 5 M HCl and sterilized by autoclaving at 121 °C.
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After cooling, 10 ml of 50 g L-1 MgSO4.7H2O solution was added to the medium.
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Wheat chaff was obtained from a local supplier (Country Link Rural Supplies,
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Lara, Victoria 3212, Australia). The fungal strains were subcultured on malt extract
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agar (Oxoid) plates incubated at 25 °C. Hyphal mat plugs from the malt extract plates
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were used to inoculate 50 ml minimal media containing 4% (w/v) wheat chaff in 250
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ml Erlenmeyer flasks. The wheat chaff was not treated prior to inoculation. Flasks were incubated at 25 °C on an orbital shaker set at 180 rpm. After 11 days there was visible hyphal mass in the flasks and fungal growth appeared to have softened and partially digested but not completely solubilized the wheat chaff.
2.1.2 Crude enzyme preparation A portion (20 ml) of the supernatant (included hyphal fragments) from the 250 ml
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flasks was used to inoculate 750 ml of minimal media containing 4% (w/v) wheat
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chaff in 2 l Erlenmeyer flasks. The flasks were incubated at 25 °C on an orbital shaker
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set at 120 rpm for 16 days. Cultures were sieved through 2 mm mesh to remove larger
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solids, and then centrifuged at 4 °C for 15 min at 12,227g to pellet smaller solids and
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fungal hyphae. The supernatant, containing the extracellular fungal enzymes, was
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decanted and stored at 4 °C.
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123 2.2 Constituent analysis
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Acid detergent lignin (ADL), acid detergent fibre (ADF) and neutral detergent
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fibre (NDF) were determined on triplicate samples by Agrifood Technology Pty Ltd,
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Werribee, VIC 3000, Australia, using the methods supplied (ANKOM Technology).
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From the NDF (composed primarily of cellulose, hemicellulose and lignin) and ADF
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(composed primarily of cellulose and lignin), hemicellulose was obtained, whereas
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cellulose was obtained from the ADF and ADL. The raw wheat chaff contained
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hemicelluloses (27.8 ± 0.3%), cellulose (21.4 ± 0.5%) and lignin (1.7 ± 0.3%) on a
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dry matter basis.
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2.3 Treatment of wheat chaff
The experimental design is outlined in Fig. 1. The wheat chaff consisted of
roughly chopped pieces (~ 1 cm). Briefly, the wheat chaff (8% w/v) was immersed in
an aqueous solution of sodium acetate buffer (2% w/v, pH 5) in a 250 ml volume
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conical flask. The samples were then microwave treated on high power (1 min) and
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immediately cooled in iced-water. Ultrasound pre-treatment was performed using a
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Blackstone-Ney Ultrasonics, MultiSONIK-2TM 7 frequency, Ultrasonic Generator
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(Blackstone-Ney Ultrasonics Inc, Jamestown, New York, USA, 900 cm2 flat
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transducer plate, programmable duty cycle, output power and frequency) at 40 kHz,
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0.5 W cm-2, 35 °C, for 10 min followed immediately by 270 kHz, 0.5 W cm-2, 35 °C
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for a further 10 min. To obtain a balance between possible loss of enzyme activity and
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improved mass transfer at high temperature, a temperature of 35 °C was used
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throughout pre-treatment and enzymatic hydrolysis. A short ultrasonic pre-treatment
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(5−10 min) has been shown to be sufficiently long to modify lignocellulosic
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substrates (Kadimaliev et al., 2003; Li et al., 2010; Chen et al., 2011) and increase the
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biomass biodegradation (Kadimaliev et al., 2003). Hence, a 10 min treatment time
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was selected for both the initial and second steps of the sequential ultrasonic treatment.
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Such a brief treatment time is also considered a practical time limit for a batch process
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that is intended to be ultimately converted into a continuous process (Ashokkumar et
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al., 2010; Lee et al., 2011).
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To assess the efficacy of pre-treatment on biodegradation, all pre-treated samples
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were subsequently inoculated independently with the crude enzyme extract (100 ml)
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obtained from either Trametes sp. or Phanerochaete chrysosporium. An aliquot (~1
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ml) of the aqueous phase was immediately removed (0 h), and then kept at 4 °C, prior
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to further analysis. The remaining wheat chaff suspension was further incubated at 35 °C for 72 h. During this time they were subjected to acoustic pulses of 80 kHz (50%
power, 0.25 W cm-2) for 1 min every 30 min. The medium frequency pulsed
ultrasound (80 kHz) was applied to enhance the activity of the enzymes, by
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facilitating mass transfer. Aliquots (~1 ml) of the aqueous phase were removed at 2, 4,
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8, 24, 48 and 72 h, and treated as per the 0 h aliquot. After 72 h incubation, the
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remaining solids were filtered (metal sieve), rinsed well with deionized water, frozen
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and freeze-dried. Control samples (i.e. no pre-treatment) were incubated
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simultaneously under otherwise equivalent conditions. All experiments were
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performed in duplicate.
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The volatile compounds generated during enzymatic degradation of lignocellulose
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in wheat chaff were analyzed by Agilent GCMS (GCMS- 6890N model GC and
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5975B model MSD) system equipped with the combiPAL robotic autosampler (CTC
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Analytics AG, Switzerland), according to Ying et al. (2010). The aqueous sample
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(1.5–2.0 g), taken at pre-determined times, was mixed with an internal standard
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solution (containing 2-methyl-3-heptanone and 2-ethyl butyric acid in methanol) in a
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22 ml screw cap headspace vial. Volatile metabolites in the sample vial were
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extracted/concentrated on the solid-phase micro-extraction (SPME) fibre
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(carboxen/polydimethylsiloxane, CAR/PDMS; 85 μm film thickness; 10 mm long;
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Supelco, Bellefonte PA 16823, USA) for 30 min at 50 oC. After 30 min of SPME
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sampling of volatiles, the SPME fibre was retracted back into the needle sleeve and
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removed from the vial. SPME sampled volatiles were thermally desorbed in the GC inlet (PTV inlet- 260 oC, set in splitless mode, splitless time 1 min) and
chromatographed on a VF-WAXms column (30 m x 0.32 mm x 1.0 μm; Varian Inc, Mulgrave VIC 3170. Australia) using a temperature gradient of 35 to 225 oC at 10 oC min-1 with initial and final hold times of 3 and 10 min, respectively. Eluted
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compounds were analyzed by MS detector (MSD). MSD conditions were as follows:
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capillary direct interface temperature, 260 °C; ionization energy, 70 eV; mass range,
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30−300 amu; scan rate, 5 scans s-1. Duplicate analyses were performed on each
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sample (the variation in duplicate analysis was less than 5%).
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Compounds were identified by comparison of mass spectra with the spectra in the NIST08 database (National Institute of Standards Technology mass spectral
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search program; NIST, United States of America) and linear retention indices
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(determined using a set of saturated alkanes C7 to C22). Quantitative analysis was
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performed using the internal standard.
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Samples were visualised using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM).
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Fluorescence of the freeze-dried wheat chaff solids was visualized using an Argon laser with excitation at 488 nm.
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2.4.1 Confocal laser scanning microscopy
2.4.2 Scanning electron microscopy
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Freeze-dried wheat chaff was mounted directly on double-sided, conductive carbon
tape affixed to the aluminium, SEM sample stub. A small portion of silver paste was used to secure the sample and to aid conductivity during scanning. Prepared samples
were provided with a thin, 1 nm platinum-palladium metal coating (in the Cressington 208HR sputter coater) to enhance image scanning. The samples were imaged in the
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Hitachi S-4300 SE/N Field Emission, Schottky Scanning Electron Microscope,
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operated at a 1.2 kV accelerating voltage, to provide surface characteristics.
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3. Results and Discussion
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The physical effects of ultrasound were observed in the disruption of the
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treated wheat chaff (Fig. 2-4). GCMS of headspace volatiles confirmed that numerous
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compounds including phenolic compounds, alcohols, acids and esters were obtained
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due to the effects of the ultrasound pre-treatment followed by enzymatic treatment of
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the wheat chaff (Fig. 5).
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Various components, such as wax, lignin, cellulose, phenolics, chlorophyll and
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amino acids in the wheat chaff display fluorescence. A decrease in fluorescence may
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therefore be linked to an increase in degradation of the wheat chaff. There was a
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relative decrease in fluorescence of the wheat chaff samples following pre-treatment
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by ultrasound, as visualized by CLSM (Fig. 2). There was a noticeable decrease in
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relative fluorescence in the ultrasonically pre-treated wheat chaff samples incubated
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without addition of the enzyme extract (Controls - Compare Fig. 2A with 2B).When
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wheat chaff pre-treated by ultrasound was inoculated with the enzyme extract
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obtained from Phanerochaete chrysosporium, a further decrease in the relative fluorescence was observed (compare Fig. 2B with 2D). Similar results were found for the ultrasonically pre-treated wheat chaff inoculated with the enzyme extract obtained from Trametes sp. A comparison between pre-treatment with ultrasound alone (Fig. 2B) and a sequence of microwave then ultrasound (Fig. 2F) indicated that addition of
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the microwave treatment did not assist in the biodegradation of wheat chaff. This
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perhaps suggests that irradiation may have partially destroyed the natural microflora
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inherently associated with the wheat chaff, which had contributed to its degradation.
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This could explain why sequential microwave and ultrasonic pre-treatment (Fig. 2E)
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was not as effective as ultrasonic pre-treatment alone (Fig. 2A). In addition,
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microwave heating may have caused condensation reactions to produce structures that
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resist enzymatic degradation. Other workers (Chen et al., 2011; Binod et al., 2012)
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found using acid or alkali in combination with microwaves, was more effective for
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delignification than microwaving alone. The apparent reason for the difference
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between our work and that of others (Chen et al., 2011; Binod et al., 2012), is because
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in our work the indigenous microflora presumably contributed to the enzyme
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degradation process whereas when an acid or alkali pre-treatment was used, the
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indigenous microflora were inactivated.
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The lack of visible structural detail of the wheat chaff pre-treated by ultrasound alone, as obtained by confocal imaging using fluorescence (Fig. 2), and the relatively
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greater increase in visual browning of the aqueous phase of these samples following
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subsequent enzymatic hydrolysis suggest that the sequential ultrasonic pre-treatment
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enhanced the enzyme accessibility to the lignocellulose matrix.
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Removal/redistribution of the wax by ultrasonication was evident in the SEM
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images (Compare Fig. 3A with 3B, and 3C with 3D). Lipophilic materials and surface
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irregularities attract the attachment of cavitation bubbles, thus, maximizing the effect
of bubble-induced micro-jetting towards these points and maximizing removal of the
lipophilic material and damage to the surface substructure (Leighton, 1994; Gale & Busnaina, 1995). The dense network of platelet structure of epicuticular wax on the
wheat leaf blade (Koch et al., 2006) could potentially have attracted cavitation bubbles in a similar way. In addition to removal of the wax, ultrasonication caused small implosions
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(Compare Fig. 3C with 3D), and pitting (Compare Fig. 3E with 3F) on the surfaces of
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the plant material, due to degradation of the cell wall components. Enhanced
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visualization of the underlying cellulose fibrils after ultrasound by SEM (Compare Fig.
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3G with 3H) provided further evidence that ultrasound had caused erosion of the
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wheat chaff. Taken together the CLSM and SEM data suggested that ultrasonic pre-treatment
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disrupted the lignocellulose matrix and stripped away/degraded the waxy surface
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components. Ultrasound-induced disruptive effects to plant material have been
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previously observed (Sun & Tomkinson 2002; Yachmenev, Condon, Klasson, &
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Lambert, 2009). These structural alterations are expected to facilitate the rate of
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enzymatic hydrolysis of plant tissue through increasing substrate accessibility. The
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observation that wax associated with the wheat chaff were removed by ultrasonic pre-
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treatment is in line with the observations of others who showed that the removal of
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hydrophobic non-cellulosics (i.e. waxes, pectins, proteins and fats) could be facilitated
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with use of ultrasound in combination with enzyme processing (Yachmenev et al.,
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2004) . Alteration of the epicuticular wax ultrastructure of grape berry fruits was
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recently observed by Fava et al. (2011).
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3.2 Headspace volatiles
To ascertain the effect of pre-treatment on biodegradation of wheat chaff by white
rot enzyme extracts GC profiles of the headspace volatiles from the hydrolyzed pretreated samples was obtained. Typical GC profiles of the headspace volatiles
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produced after ultrasonic pre-treatment of wheat chaff and its subsequent incubation
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for 72 h, with crude white rot fungal enzyme extracts, are shown in Fig. 4. Numerous
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compounds including phenolic compounds, alcohols, acids and esters, associated with
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degradation of the wheat chaff were identified after enzyme hydrolysis. The phenolic-
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derived products are likely to arise from both degradation of lignin and also the
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enzymatic hydrolysis of lignocellulose-derived components during fermentation. The
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major lignin degradation products, such as variously substituted guaiacols and
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syringols (Martínez et al., 2005), have relatively high boiling points and are more
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polar, and for these reasons were not detected in the headspace profile using the GC
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conditions applied. Moreover, liginases produced by white rot fungi such as P.
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chrysosporium can cause demethylation of guaiacyl and syringyl derivatives (Karsten,
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Tien, Kalyanararnan, & Kirk, 1985; Huynh & Crawford, 1985) and additionally the
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higher frequency of 270 kHz we used may have generated free radicals that acted
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preferentially on the guaiacyl and syringyl compounds. The alcohols (ethanol, 2-
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pentanol, 3-methyl butanol, 1-pentanol, 1-hexanol), acids (acetic, propanoic, hexanoic,
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octanoic and nonanoic acids) and ester compounds (methyl acetate, ethyl acetate)
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identified in the headspace analysis are typical products derived from microbial
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fermentation of cellulose and hemicelluloses (Ashraf, Linforth, Bealin-Kelly, Smart,
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& Taylor, 2010). The most likely reason that ethanol and other products typical of a
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fermentation process were found is that the microflora associated with the wheat chaff
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(and possibly also from the environment) grew on the more readily biodegradable
components that had been released from the wheat chaff (e.g. soluble carbohydrates) and either solubilized in the presence of water or released by the ultrasonic pretreatment. Some fatty acids (e.g. hexanoic acid, octanoic acid, nonanoic acid) were also present in the headspace. These compounds could arise from degradation of the
long chain fatty acids present in the plant cuticle and/or cell wall.
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3.3.1 Quantification of volatile phenolic-derived compounds
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A range of phenolic-derived compounds, including benzaldehyde, phenyl methyl acetate, phenyl ethyl acetate, benzyl alcohol, phenylmethyl alcohol and phenol, were
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quantified (Fig. 5). The type and concentration of the volatiles produced depended on
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the type of pre-treatment that had been applied to the wheat chaff.
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Generally, greater levels of phenolic-derived volatiles were detected for wheat
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chaff pre-treated by ultrasound, compared to pre-treatment by microwave, combined
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microwave and ultrasound, or control samples (i.e. incubation without any physical
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treatment, under otherwise under comparable conditions). The inclusion of a
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microwave pre-treatment, with or without ultrasound, generally resulted in lower
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levels of phenolic-derived products. The detrimental effect of a microwave treatment
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may be related to the destructive effect of microwaves on the natural microflora
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inherently associated with the wheat chaff, which assisted in its biodegradation. The
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effect of delignification was less pronounced when the ultrasonic pre-treatment was
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followed by hydrolysis with the enzyme extract obtained from Trametes sp. (Fig. 5A)
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compared to Phanerochaete chrysosporium (Fig. 5B). These differences may be
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related to the different types and activities of the enzymes produced by the white rot
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fungi. Multiple laccase genes are recognised in the lignin-degrading basidiomycetes belonging to the genera Trametes, and several genes proposed to encode lignin peroxidase have been reported in T. versicolor (Jönsson & Nyman, 1992). In contrast,
P. chrysosporium is widely cited as a white rot fungus that does not produce laccase,
but its production of lignin peroxidase and manganese peroxidase, is well established (Hatakka, 1994).
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4. Conclusion
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Modification of wheat chaff by low intensity sequential low and medium frequency ultrasound enhanced disruption of the lignocellulose matrix and may be
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recommended for accelerating subsequent enzymatic degradation of lignocellulosic
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substrates. The fungus from which the hydrolytic enzymes were derived influenced
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the type and quantity of phenolic-derived volatiles generated. Microwave pre-
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treatment led to less phenolic-derived volatiles being produced, suggesting that the
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microbial consortia inherently associated with the wheat chaff also contributed to its
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biodegradation. Further study is needed to optimize conditions with respect to
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treatment time and temperature, ultrasonic frequencies and power intensities for
351
degrading the wheat chaff and recovering the myriad of potentially useful products
352
that can be isolated from lignocellulose products.
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353 Acknowledgements
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The authors would like to acknowledge Piotr Swiergon for his advice and guidance in
356
the using the Blackstone-Ney Ultrasonics, MultiSONIK-2TM 7 frequency, Ultrasonic
357
Generator, and Colin Veitch and Margaret Pate (CSIRO Materials Science and
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Engineering) for the SEM pictures.
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Ying, D., Sanguansri, L., Weerakkody, R., Singh, T. K., Leischtfeld, S. F.,
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Gantenbein-Demarchi, C., & Augustin, M. A. (2010). Tocopherol and ascorbate
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have contrasting effects on the viability of microencapsulated Lactobacillus
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rhamnosus GG. Journal of Agricultural and Food Chemistry, 59, 10556 – 10563.
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Zakzeski, J., Bruijnincx, P. C. A., Jongerius, A. L., & Weckhuysen, B. M. (2010). The
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Chemical Reviews, 110, 3552 – 3599.
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Figure Captions
479 Fig. 1. Outline of experimental design. P = P. chrysosporium. T = Trametes sp. US =
481
ultrasound
482
Fig. 2. Fluorescence at 488 nm of freeze-dried wheat chaff solids following pre-
483
treatment and incubation for 72 h at 35 °C. The fungal extract was obtained from P.
484
chrysosporium. All images 817 x 817 μm. Control A & B, enzyme treated C & D and
485
E & F with microwave and enzyme). A , C & E no ultrasonic treatment and B, D and
486
F with ultrasonic treatment. Magnification bar 250 μm.
487
Fig. 3. SEM micrographs of the lower surface (A - D), and upper surface (E & F), of
488
wheat chaff leaves and surface of stem (G& H). Wheat chaff (8% w/v solids in 2%
489
sodium acetate buffer, pH 5) was incubated at 35 °C, in the absence (A, C, E, G) and
490
presence (B, D, F, H) of ultrasound (40 kHz, 10 min). Magnification bar A & B 250
491
μm and C - G 25 μm.
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Fig. 4. Typical GC profile of volatiles produced from wheat chaff after sequential
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ultrasonication (40 kHz, 10 min/270 kHz, 10 min) and incubation (72 h at 35 °C) with
498
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phenylmethyl acetate, phenylethyl acetate) produced from wheat chaff after sequential
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ultrasonication (40 kHz, 10 min/270 kHz, 10 min, microwave at 650 W and sequential
501
ultrasonication, microwave alone) and incubation (72 h at 35 °C) with enzyme
502
extracts obtained from Trametes sp. (A) and P. chrysosporium (B). Values represent
494 495 496 497
enzyme extracts obtained from P. chrysosporium and Trametes sp. Internal standards
IS#1 2-methyl-3-heptanone, IS#2 2-ethyl butyric acid.
Fig. 5. Quantitative data on dominant headspace phenolic-derived compounds above treatment liquors (benzaldehyde, benzyl alcohol, phenol, phenylethyl alcohol,
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concentrations in the treated liquor derived from GCMS headspace gas spectra using
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equipment software internal reference standards. Internal standards IS#1 2-methyl-3-
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heptanone, IS#2 2-ethyl butyric acid.
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Highlights: Ultrasound pre-treatment of wheat chaff facilitates white rot enzyme digestion Ultrasound improves increases enzyme accessibility to the cellulose fibrils P. chrysosporium degraded uultrasound-treated chaff more than Trametes sp.
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S0144-8617(14)00385-3 http://dx.doi.org/doi:10.1016/j.carbpol.2014.04.028 CARP 8789
To appear in: Received date: Revised date: Accepted date:
27-9-2013 31-3-2014 8-4-2014
Please cite this article as: Oliver, C. M., Mawson, R., Melton, L. D., Dumsday, G., Welch, J., Singh, P. S., Tanoj K., & Augustin, M. A.,Sequential low and medium frequency ultrasound assists biodegradation of wheat chaff by white rot fungal enzymes, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.04.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sequential low and medium frequency ultrasound assists
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biodegradation of wheat chaff by white rot fungal enzymes
Christine M. Olivera, Raymond Mawsona, Laurence D. Meltonb, Geoff Dumsdayc,
CSIRO Animal, Food and Health Sciences, 671 Sneydes Road, Werribee, VIC 3030,
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a
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Jessica Welchc, Peerasak Sanguansria Tanoj K. Singha, Mary Ann Augustina*
Australia
Food Science, School of Chemical Sciences, University of Auckland, Private Bag
an
b
92019, Auckland, New Zealand c
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CSIRO Material Science and Engineering, Normanby Road, Clayton, VIC 3168,
d
Australia
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*Corresponding author. Address: CSIRO Animal, Food and Health Sciences, 671
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Sneydes Rd, Werribee, VIC 3030, Australia. Tel.: +61 3 9731 3486; fax +61 3 9731 3201.
E-mail address: [email protected]
1
Page 1 of 30
Abstract The consequences of a short (10 min per frequency) ultrasonic pre-
2
treatment using low (40 kHz), medium (270 kHz), or sequential low and medium
3
frequency (40 kHz followed by 270 kHz), on the degradation of wheat chaff (8 g 100
4
ml-1 acetate buffer, pH 5) were evaluated. In addition, the effects of the ultrasonic
5
treatment on the degradation of the wheat chaff when subsequently exposed to
6
enzyme extracts from two white rot fungi (Phanerochaete chrysosporium and
7
Trametes sp.) were investigated. Pre-treatment by sequential low and medium
8
frequency ultrasound had a disruptive effect on the lignocellulosic matrix. Analysis of
9
the phenolic-derived volatiles after enzymatic hydrolysis showed that biodegradation
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with the enzyme extract obtained from Phanerochaete chrysosporium was more
11
pronounced compared to that of the Trametes sp. The efficacy of the ultrasonic pre-
12
treatment was attributed to increased enzyme accessibility of the cellulose fibrils due
13
to sonication-induced disruption of the plant surface structure, as shown by changes in
14
the microstructure.
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Keywords Ultrasound, White rot fungal enzymes, Phanerochaete chrysosporium,
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Trametes sp., Wheat chaff, Biodegradation
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18 19
1.
Introduction
20 Lignocellulosic biomass is an abundant and renewable source of fuel, energy and organic chemicals and has potential application as an animal feed (Sun, 2010). Pre-
23
treatment of lignocellulosic biomass is necessary to alter its physical and chemical
24
structure for improved cellulose/hemicellulose accessibility (Weng, Li, Bonawitz, &
25
Chapple, 2008; Zakzeski, Bruijnincx, Jongerius, & Weckhuysen, 2010; Volynets &
26
Dahman, 2011). Current pre-treatment methods used to degrade lignocellulosic
27
biomass involve the utilization of harsh chemicals (e.g. strong bases, concentrated
28
sulphuric acid, oxidizing agents) (da Costa Sousa, Chundawat, Balan, & Dale, 2009)
29
or energy-intensive technologies such as steam explosion (Josefsson, Lennholm, &
30
Gellerstedt, 2001; Hana, Deng, Zhang, Bicho, & Wue, 2010). Biological pre-
31
treatment of lignocellulosic biomass is an alternative more environmentally-friendly
32
approach (Leonowicz et al., 1999; Sánchez, 2009). The most promising
33
microorganisms for biological pre-treatment are basidomycetes, which degrade lignin
34 35 36 37 38
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to varying extents depending on the type of fungi and fermentation conditions used (Dinis et al., 2009). These fungi secrete one or more of three extracellular enzyme
complexes that are fundamentally important for lignin degradation, predominantly lignin peroxidase, manganese peroxidase, and laccase (Hatakka, 1994; Jönsson &
Nyman, 1992). Even so, biodegradation of lignin by white rot fungi alone is itself an
39
impractically slow process. Alternative pre-treatment approaches, which not only
40
reduce the energy input and the use of harsh chemicals but increase the total process
41
efficiency of biomass transformation, are required.
42 43
Microwave irradiation has been investigated as an alternative means of pretreating lignocellulosic material. For example, Binod et al. (2012) showed that
3
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microwave-based alkali pre-treatment of sugarcane baggase removed external fibres,
45
increasing the accessibility of the cellulose to enzymes. Chen, Tu & Sheen (2011)
46
found that dilute acid pre-treatment of ground sugarcane bagasse using microwave
47
heating markedly destroyed the lignocellulose structure, causing swelling and
48
fragmentation of the bagasse particles. The ability of microwave to facilitate the
49
extraction of soluble phenolic compounds from plant material has also been reviewed
50
(Routray, & Orsat, 2012).
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Ultrasound has been used successfully in conventional processes of desizing,
52
extraction and wool wax scouring (Yachmenev, Bertoniere, & Blanchard, 2001;
53
Yachmenev, Blanchard, & Lambert, 2004; Hurren, Zhang, Liu, & Wang, 2006).
54
Typically, in such applications, mechanically pre-comminuted material is placed in a
55
medium containing either strong acid or alkali and subjected to a single low frequency
56
over the range 20−40 kHz. Sun & Tomkinson (2002) demonstrated ultrasound (20
57
kHz) assisted alkali-dissolution of ground wheat straw. Similarly Velmurugan &
58
Muthukumar (2001) applied ultrasonication (24 kHz) to assist alkali delignification of
59
powdered sugarcane bagasse by increasing surface oxidation of lignin and
61 62 63 64
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hemicellulose. Li, Sun, & Sun (2010) showed that ultrasound treatment (35 kHz) of ball-milled, dewaxed bamboo culms increased the partitioning of lignin into ethanol-
soluble extracts. Pre-treatment of sawdust with ultrasound (22 kHz) enhanced lignin
degradation during subsequent solid-phase cultivation of Panus (Lenitnus) tigrinus (Kadimaliev, Revin, Atykyan, & Samuilov, 2003). It has also been established that
65
ultrasound in combination with appropriate solvents can be used to recover lignin
66
from hemicelluloses (García, Alriols, Llano-Ponte, & Labidi, 2011).
67
Ultrasound also has the potential to increase the activities of enzymatic processes.
68
For example, ultrasound (16 and 20 kHz, up to 3 W cm-2) has been shown to enhance
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the enzymatic bio-scouring of cotton (Yachmenev et al., 2004). While the disruptive
70
effects of cavitation from low frequency ultrasound (20−100 kHz) physically tease the
71
plant material apart and increase accessibility to the underlying cellulose, higher
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frequencies (>100 kHz) increase free radical generation and therefore may be
73
expected to sonochemically oxidize components (Ashokkumar et al., 2008).
The primary objective of this work was to evaluate the impact of low and medium
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frequency ultrasound on wheat chaff and its subsequent biodegradation by enzyme
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extracts obtained from cultivation of two white rot fungi, Trametes sp. and
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Phanerochaete chrysosporium. It was hypothesized that the use of a combination of
78
frequencies would be more effective than a single frequency for disrupting plant
79
biomass, thereby improving enzyme access to the underlying cellulose. The
80
justification for using 40 kHz as the initial pre-treatment frequency in the sequential
81
process presented here was based on the scouring of wool wax, where 35–45 kHz was
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found to be optimal for cracking the waxy layer and stripping away the scaly surface
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(Hurren et al., 2006). Additionally targeting of the cracks in the waxy cuticle and
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potential lifting of the waxy surface by micro-streaming could be expected. The
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second frequency of 270 kHz was selected to provide enhanced oxidizing potential (Ashokkumar et al., 2008) and specifically erode the closed lip crevice of the microscopic respiratory stomata, and thereby enhance accessibility of enzymes to the internal tissue structure. Based on previous literature findings using microwave (Chen
et al., 2011; Binod et al., 2012), a brief high temperature heat treatment of the wheat
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chaff prior to ultrasonic pre-treatment was also evaluated for its ability to enhance
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sonication-induced disruption of the lignocellulosic material and subsequent
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enzymatic hydrolysis.
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2. Materials and Methods
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2.1 Growth of the fungal isolates and preparation of the enzyme extracts
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The Trametes sp. strain 2403 and Phanerochaete chrysosporium strain 16543
were obtained from the CSIRO Collection of Wood Decaying Fungi. The medium
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used to grow the fungal isolates contained (per litre): 3 g NaNO3; 1 g K2HPO4; 0.5 g
101
KCl; 0.5 g yeast extract (BD Bacto) and 0.01 g FeSO4.7H2O. After formulation the
102
medium was adjusted to pH 5 using 5 M HCl and sterilized by autoclaving at 121 °C.
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After cooling, 10 ml of 50 g L-1 MgSO4.7H2O solution was added to the medium.
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Wheat chaff was obtained from a local supplier (Country Link Rural Supplies,
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Lara, Victoria 3212, Australia). The fungal strains were subcultured on malt extract
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agar (Oxoid) plates incubated at 25 °C. Hyphal mat plugs from the malt extract plates
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were used to inoculate 50 ml minimal media containing 4% (w/v) wheat chaff in 250
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ml Erlenmeyer flasks. The wheat chaff was not treated prior to inoculation. Flasks were incubated at 25 °C on an orbital shaker set at 180 rpm. After 11 days there was visible hyphal mass in the flasks and fungal growth appeared to have softened and partially digested but not completely solubilized the wheat chaff.
2.1.2 Crude enzyme preparation A portion (20 ml) of the supernatant (included hyphal fragments) from the 250 ml
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flasks was used to inoculate 750 ml of minimal media containing 4% (w/v) wheat
118
chaff in 2 l Erlenmeyer flasks. The flasks were incubated at 25 °C on an orbital shaker
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set at 120 rpm for 16 days. Cultures were sieved through 2 mm mesh to remove larger
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solids, and then centrifuged at 4 °C for 15 min at 12,227g to pellet smaller solids and
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fungal hyphae. The supernatant, containing the extracellular fungal enzymes, was
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decanted and stored at 4 °C.
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123 2.2 Constituent analysis
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Acid detergent lignin (ADL), acid detergent fibre (ADF) and neutral detergent
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fibre (NDF) were determined on triplicate samples by Agrifood Technology Pty Ltd,
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Werribee, VIC 3000, Australia, using the methods supplied (ANKOM Technology).
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From the NDF (composed primarily of cellulose, hemicellulose and lignin) and ADF
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(composed primarily of cellulose and lignin), hemicellulose was obtained, whereas
131
cellulose was obtained from the ADF and ADL. The raw wheat chaff contained
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hemicelluloses (27.8 ± 0.3%), cellulose (21.4 ± 0.5%) and lignin (1.7 ± 0.3%) on a
133
dry matter basis.
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2.3 Treatment of wheat chaff
The experimental design is outlined in Fig. 1. The wheat chaff consisted of
roughly chopped pieces (~ 1 cm). Briefly, the wheat chaff (8% w/v) was immersed in
an aqueous solution of sodium acetate buffer (2% w/v, pH 5) in a 250 ml volume
140
conical flask. The samples were then microwave treated on high power (1 min) and
141
immediately cooled in iced-water. Ultrasound pre-treatment was performed using a
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Blackstone-Ney Ultrasonics, MultiSONIK-2TM 7 frequency, Ultrasonic Generator
143
(Blackstone-Ney Ultrasonics Inc, Jamestown, New York, USA, 900 cm2 flat
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transducer plate, programmable duty cycle, output power and frequency) at 40 kHz,
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0.5 W cm-2, 35 °C, for 10 min followed immediately by 270 kHz, 0.5 W cm-2, 35 °C
146
for a further 10 min. To obtain a balance between possible loss of enzyme activity and
147
improved mass transfer at high temperature, a temperature of 35 °C was used
148
throughout pre-treatment and enzymatic hydrolysis. A short ultrasonic pre-treatment
149
(5−10 min) has been shown to be sufficiently long to modify lignocellulosic
150
substrates (Kadimaliev et al., 2003; Li et al., 2010; Chen et al., 2011) and increase the
151
biomass biodegradation (Kadimaliev et al., 2003). Hence, a 10 min treatment time
152
was selected for both the initial and second steps of the sequential ultrasonic treatment.
153
Such a brief treatment time is also considered a practical time limit for a batch process
154
that is intended to be ultimately converted into a continuous process (Ashokkumar et
155
al., 2010; Lee et al., 2011).
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To assess the efficacy of pre-treatment on biodegradation, all pre-treated samples
157
were subsequently inoculated independently with the crude enzyme extract (100 ml)
158
obtained from either Trametes sp. or Phanerochaete chrysosporium. An aliquot (~1
159
ml) of the aqueous phase was immediately removed (0 h), and then kept at 4 °C, prior
161 162 163
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to further analysis. The remaining wheat chaff suspension was further incubated at 35 °C for 72 h. During this time they were subjected to acoustic pulses of 80 kHz (50%
power, 0.25 W cm-2) for 1 min every 30 min. The medium frequency pulsed
ultrasound (80 kHz) was applied to enhance the activity of the enzymes, by
164
facilitating mass transfer. Aliquots (~1 ml) of the aqueous phase were removed at 2, 4,
165
8, 24, 48 and 72 h, and treated as per the 0 h aliquot. After 72 h incubation, the
166
remaining solids were filtered (metal sieve), rinsed well with deionized water, frozen
167
and freeze-dried. Control samples (i.e. no pre-treatment) were incubated
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168
simultaneously under otherwise equivalent conditions. All experiments were
169
performed in duplicate.
170 2.4 Analysis of headspace volatiles
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The volatile compounds generated during enzymatic degradation of lignocellulose
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173
in wheat chaff were analyzed by Agilent GCMS (GCMS- 6890N model GC and
175
5975B model MSD) system equipped with the combiPAL robotic autosampler (CTC
176
Analytics AG, Switzerland), according to Ying et al. (2010). The aqueous sample
177
(1.5–2.0 g), taken at pre-determined times, was mixed with an internal standard
178
solution (containing 2-methyl-3-heptanone and 2-ethyl butyric acid in methanol) in a
179
22 ml screw cap headspace vial. Volatile metabolites in the sample vial were
180
extracted/concentrated on the solid-phase micro-extraction (SPME) fibre
181
(carboxen/polydimethylsiloxane, CAR/PDMS; 85 μm film thickness; 10 mm long;
182
Supelco, Bellefonte PA 16823, USA) for 30 min at 50 oC. After 30 min of SPME
183
sampling of volatiles, the SPME fibre was retracted back into the needle sleeve and
185 186 187 188
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removed from the vial. SPME sampled volatiles were thermally desorbed in the GC inlet (PTV inlet- 260 oC, set in splitless mode, splitless time 1 min) and
chromatographed on a VF-WAXms column (30 m x 0.32 mm x 1.0 μm; Varian Inc, Mulgrave VIC 3170. Australia) using a temperature gradient of 35 to 225 oC at 10 oC min-1 with initial and final hold times of 3 and 10 min, respectively. Eluted
189
compounds were analyzed by MS detector (MSD). MSD conditions were as follows:
190
capillary direct interface temperature, 260 °C; ionization energy, 70 eV; mass range,
191
30−300 amu; scan rate, 5 scans s-1. Duplicate analyses were performed on each
192
sample (the variation in duplicate analysis was less than 5%).
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193
Compounds were identified by comparison of mass spectra with the spectra in the NIST08 database (National Institute of Standards Technology mass spectral
195
search program; NIST, United States of America) and linear retention indices
196
(determined using a set of saturated alkanes C7 to C22). Quantitative analysis was
197
performed using the internal standard.
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199
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202
Samples were visualised using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM).
207 208 209 210 211 212 213
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Fluorescence of the freeze-dried wheat chaff solids was visualized using an Argon laser with excitation at 488 nm.
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2.4.1 Confocal laser scanning microscopy
2.4.2 Scanning electron microscopy
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Freeze-dried wheat chaff was mounted directly on double-sided, conductive carbon
tape affixed to the aluminium, SEM sample stub. A small portion of silver paste was used to secure the sample and to aid conductivity during scanning. Prepared samples
were provided with a thin, 1 nm platinum-palladium metal coating (in the Cressington 208HR sputter coater) to enhance image scanning. The samples were imaged in the
214
Hitachi S-4300 SE/N Field Emission, Schottky Scanning Electron Microscope,
215
operated at a 1.2 kV accelerating voltage, to provide surface characteristics.
216 217
3. Results and Discussion
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Page 10 of 30
The physical effects of ultrasound were observed in the disruption of the
219
treated wheat chaff (Fig. 2-4). GCMS of headspace volatiles confirmed that numerous
220
compounds including phenolic compounds, alcohols, acids and esters were obtained
221
due to the effects of the ultrasound pre-treatment followed by enzymatic treatment of
222
the wheat chaff (Fig. 5).
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223 3.1 Microscopy
Various components, such as wax, lignin, cellulose, phenolics, chlorophyll and
an
226
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amino acids in the wheat chaff display fluorescence. A decrease in fluorescence may
228
therefore be linked to an increase in degradation of the wheat chaff. There was a
229
relative decrease in fluorescence of the wheat chaff samples following pre-treatment
230
by ultrasound, as visualized by CLSM (Fig. 2). There was a noticeable decrease in
231
relative fluorescence in the ultrasonically pre-treated wheat chaff samples incubated
232
without addition of the enzyme extract (Controls - Compare Fig. 2A with 2B).When
233
wheat chaff pre-treated by ultrasound was inoculated with the enzyme extract
235 236 237 238
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obtained from Phanerochaete chrysosporium, a further decrease in the relative fluorescence was observed (compare Fig. 2B with 2D). Similar results were found for the ultrasonically pre-treated wheat chaff inoculated with the enzyme extract obtained from Trametes sp. A comparison between pre-treatment with ultrasound alone (Fig. 2B) and a sequence of microwave then ultrasound (Fig. 2F) indicated that addition of
239
the microwave treatment did not assist in the biodegradation of wheat chaff. This
240
perhaps suggests that irradiation may have partially destroyed the natural microflora
241
inherently associated with the wheat chaff, which had contributed to its degradation.
242
This could explain why sequential microwave and ultrasonic pre-treatment (Fig. 2E)
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was not as effective as ultrasonic pre-treatment alone (Fig. 2A). In addition,
244
microwave heating may have caused condensation reactions to produce structures that
245
resist enzymatic degradation. Other workers (Chen et al., 2011; Binod et al., 2012)
246
found using acid or alkali in combination with microwaves, was more effective for
247
delignification than microwaving alone. The apparent reason for the difference
248
between our work and that of others (Chen et al., 2011; Binod et al., 2012), is because
249
in our work the indigenous microflora presumably contributed to the enzyme
250
degradation process whereas when an acid or alkali pre-treatment was used, the
251
indigenous microflora were inactivated.
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The lack of visible structural detail of the wheat chaff pre-treated by ultrasound alone, as obtained by confocal imaging using fluorescence (Fig. 2), and the relatively
254
greater increase in visual browning of the aqueous phase of these samples following
255
subsequent enzymatic hydrolysis suggest that the sequential ultrasonic pre-treatment
256
enhanced the enzyme accessibility to the lignocellulose matrix.
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Removal/redistribution of the wax by ultrasonication was evident in the SEM
258
images (Compare Fig. 3A with 3B, and 3C with 3D). Lipophilic materials and surface
259 260 261 262 263 264 265
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irregularities attract the attachment of cavitation bubbles, thus, maximizing the effect
of bubble-induced micro-jetting towards these points and maximizing removal of the
lipophilic material and damage to the surface substructure (Leighton, 1994; Gale & Busnaina, 1995). The dense network of platelet structure of epicuticular wax on the
wheat leaf blade (Koch et al., 2006) could potentially have attracted cavitation bubbles in a similar way. In addition to removal of the wax, ultrasonication caused small implosions
266
(Compare Fig. 3C with 3D), and pitting (Compare Fig. 3E with 3F) on the surfaces of
267
the plant material, due to degradation of the cell wall components. Enhanced
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268
visualization of the underlying cellulose fibrils after ultrasound by SEM (Compare Fig.
269
3G with 3H) provided further evidence that ultrasound had caused erosion of the
270
wheat chaff. Taken together the CLSM and SEM data suggested that ultrasonic pre-treatment
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disrupted the lignocellulose matrix and stripped away/degraded the waxy surface
273
components. Ultrasound-induced disruptive effects to plant material have been
274
previously observed (Sun & Tomkinson 2002; Yachmenev, Condon, Klasson, &
275
Lambert, 2009). These structural alterations are expected to facilitate the rate of
276
enzymatic hydrolysis of plant tissue through increasing substrate accessibility. The
277
observation that wax associated with the wheat chaff were removed by ultrasonic pre-
278
treatment is in line with the observations of others who showed that the removal of
279
hydrophobic non-cellulosics (i.e. waxes, pectins, proteins and fats) could be facilitated
280
with use of ultrasound in combination with enzyme processing (Yachmenev et al.,
281
2004) . Alteration of the epicuticular wax ultrastructure of grape berry fruits was
282
recently observed by Fava et al. (2011).
284 285 286 287 288
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3.2 Headspace volatiles
To ascertain the effect of pre-treatment on biodegradation of wheat chaff by white
rot enzyme extracts GC profiles of the headspace volatiles from the hydrolyzed pretreated samples was obtained. Typical GC profiles of the headspace volatiles
289
produced after ultrasonic pre-treatment of wheat chaff and its subsequent incubation
290
for 72 h, with crude white rot fungal enzyme extracts, are shown in Fig. 4. Numerous
291
compounds including phenolic compounds, alcohols, acids and esters, associated with
292
degradation of the wheat chaff were identified after enzyme hydrolysis. The phenolic-
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Page 13 of 30
derived products are likely to arise from both degradation of lignin and also the
294
enzymatic hydrolysis of lignocellulose-derived components during fermentation. The
295
major lignin degradation products, such as variously substituted guaiacols and
296
syringols (Martínez et al., 2005), have relatively high boiling points and are more
297
polar, and for these reasons were not detected in the headspace profile using the GC
298
conditions applied. Moreover, liginases produced by white rot fungi such as P.
299
chrysosporium can cause demethylation of guaiacyl and syringyl derivatives (Karsten,
300
Tien, Kalyanararnan, & Kirk, 1985; Huynh & Crawford, 1985) and additionally the
301
higher frequency of 270 kHz we used may have generated free radicals that acted
302
preferentially on the guaiacyl and syringyl compounds. The alcohols (ethanol, 2-
303
pentanol, 3-methyl butanol, 1-pentanol, 1-hexanol), acids (acetic, propanoic, hexanoic,
304
octanoic and nonanoic acids) and ester compounds (methyl acetate, ethyl acetate)
305
identified in the headspace analysis are typical products derived from microbial
306
fermentation of cellulose and hemicelluloses (Ashraf, Linforth, Bealin-Kelly, Smart,
307
& Taylor, 2010). The most likely reason that ethanol and other products typical of a
308
fermentation process were found is that the microflora associated with the wheat chaff
310 311 312 313 314
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(and possibly also from the environment) grew on the more readily biodegradable
components that had been released from the wheat chaff (e.g. soluble carbohydrates) and either solubilized in the presence of water or released by the ultrasonic pretreatment. Some fatty acids (e.g. hexanoic acid, octanoic acid, nonanoic acid) were also present in the headspace. These compounds could arise from degradation of the
long chain fatty acids present in the plant cuticle and/or cell wall.
315 316
3.3.1 Quantification of volatile phenolic-derived compounds
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317
A range of phenolic-derived compounds, including benzaldehyde, phenyl methyl acetate, phenyl ethyl acetate, benzyl alcohol, phenylmethyl alcohol and phenol, were
319
quantified (Fig. 5). The type and concentration of the volatiles produced depended on
320
the type of pre-treatment that had been applied to the wheat chaff.
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Generally, greater levels of phenolic-derived volatiles were detected for wheat
322
chaff pre-treated by ultrasound, compared to pre-treatment by microwave, combined
323
microwave and ultrasound, or control samples (i.e. incubation without any physical
324
treatment, under otherwise under comparable conditions). The inclusion of a
325
microwave pre-treatment, with or without ultrasound, generally resulted in lower
326
levels of phenolic-derived products. The detrimental effect of a microwave treatment
327
may be related to the destructive effect of microwaves on the natural microflora
328
inherently associated with the wheat chaff, which assisted in its biodegradation. The
329
effect of delignification was less pronounced when the ultrasonic pre-treatment was
330
followed by hydrolysis with the enzyme extract obtained from Trametes sp. (Fig. 5A)
331
compared to Phanerochaete chrysosporium (Fig. 5B). These differences may be
332
related to the different types and activities of the enzymes produced by the white rot
334 335 336 337 338
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fungi. Multiple laccase genes are recognised in the lignin-degrading basidiomycetes belonging to the genera Trametes, and several genes proposed to encode lignin peroxidase have been reported in T. versicolor (Jönsson & Nyman, 1992). In contrast,
P. chrysosporium is widely cited as a white rot fungus that does not produce laccase,
but its production of lignin peroxidase and manganese peroxidase, is well established (Hatakka, 1994).
339 340
4. Conclusion
341
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Page 15 of 30
342
Modification of wheat chaff by low intensity sequential low and medium frequency ultrasound enhanced disruption of the lignocellulose matrix and may be
344
recommended for accelerating subsequent enzymatic degradation of lignocellulosic
345
substrates. The fungus from which the hydrolytic enzymes were derived influenced
346
the type and quantity of phenolic-derived volatiles generated. Microwave pre-
347
treatment led to less phenolic-derived volatiles being produced, suggesting that the
348
microbial consortia inherently associated with the wheat chaff also contributed to its
349
biodegradation. Further study is needed to optimize conditions with respect to
350
treatment time and temperature, ultrasonic frequencies and power intensities for
351
degrading the wheat chaff and recovering the myriad of potentially useful products
352
that can be isolated from lignocellulose products.
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353 Acknowledgements
355
The authors would like to acknowledge Piotr Swiergon for his advice and guidance in
356
the using the Blackstone-Ney Ultrasonics, MultiSONIK-2TM 7 frequency, Ultrasonic
357
Generator, and Colin Veitch and Margaret Pate (CSIRO Materials Science and
359 360 361 362
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Engineering) for the SEM pictures.
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478
Figure Captions
479 Fig. 1. Outline of experimental design. P = P. chrysosporium. T = Trametes sp. US =
481
ultrasound
482
Fig. 2. Fluorescence at 488 nm of freeze-dried wheat chaff solids following pre-
483
treatment and incubation for 72 h at 35 °C. The fungal extract was obtained from P.
484
chrysosporium. All images 817 x 817 μm. Control A & B, enzyme treated C & D and
485
E & F with microwave and enzyme). A , C & E no ultrasonic treatment and B, D and
486
F with ultrasonic treatment. Magnification bar 250 μm.
487
Fig. 3. SEM micrographs of the lower surface (A - D), and upper surface (E & F), of
488
wheat chaff leaves and surface of stem (G& H). Wheat chaff (8% w/v solids in 2%
489
sodium acetate buffer, pH 5) was incubated at 35 °C, in the absence (A, C, E, G) and
490
presence (B, D, F, H) of ultrasound (40 kHz, 10 min). Magnification bar A & B 250
491
μm and C - G 25 μm.
492
Fig. 4. Typical GC profile of volatiles produced from wheat chaff after sequential
493
ultrasonication (40 kHz, 10 min/270 kHz, 10 min) and incubation (72 h at 35 °C) with
498
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480
499
phenylmethyl acetate, phenylethyl acetate) produced from wheat chaff after sequential
500
ultrasonication (40 kHz, 10 min/270 kHz, 10 min, microwave at 650 W and sequential
501
ultrasonication, microwave alone) and incubation (72 h at 35 °C) with enzyme
502
extracts obtained from Trametes sp. (A) and P. chrysosporium (B). Values represent
494 495 496 497
enzyme extracts obtained from P. chrysosporium and Trametes sp. Internal standards
IS#1 2-methyl-3-heptanone, IS#2 2-ethyl butyric acid.
Fig. 5. Quantitative data on dominant headspace phenolic-derived compounds above treatment liquors (benzaldehyde, benzyl alcohol, phenol, phenylethyl alcohol,
22
Page 22 of 30
503
concentrations in the treated liquor derived from GCMS headspace gas spectra using
504
equipment software internal reference standards. Internal standards IS#1 2-methyl-3-
505
heptanone, IS#2 2-ethyl butyric acid.
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Highlights: Ultrasound pre-treatment of wheat chaff facilitates white rot enzyme digestion Ultrasound improves increases enzyme accessibility to the cellulose fibrils P. chrysosporium degraded uultrasound-treated chaff more than Trametes sp.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5A
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Figure 5B
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