Environ. Sci. Technol. 2008, 42, 276–281
Microscale Analysis of in Vitro Anaerobic Degradation of Lignocellulosic Wastes by Rumen Microorganisms Z H E N - H U H U , †,§ S H A O - Y A N G L I U , † ZHENG-BO YUE,† LI-FENG YAN,‡ M I N G - T A O Y A N G , ‡ A N D H A N - Q I N G Y U * ,† Laboratory of Environmental Engineering, School of Chemistry, University of Science & Technology of China, Hefei, 230026, China, National Laboratory of Micro-scale Material Structure, University of Science and Technology of China, Hefei, 230026, China, School of Civil Engineering, Hefei University of Technology, Hefei, 230092, China
Received August 1, 2007. Revised manuscript received October 22, 2007. Accepted October 30, 2007.
Anaerobic degradation of lignin in waste straw by ruminal microbes was directly observed using atomic force microscope (AFM). A series of high-resolution AFM images of the straw surface in the biodegradation show that the wax flakelets and lignin granules covering the straw surface were removed by the rumen microorganisms. Such degradation resulted in an exposure of cellulose fibers located inside the straw. The appearance of holes and microfibers in fermentation reveals that tunneling might be one of the ways for rumen microorganisms to attack the straw. Increases in the atomic ratio of oxygen to carbon (O/C) and the ratio C2/C3 in C1s spectra of X-ray photoelectron spectroscopy confirm that more cellulose was exposed on the surface after the anaerobic fermentation of straw. Gas chromatography/mass spectrometry analytical results demonstrate the decomposition of lignin by rumen microorganisms. Fourier transform infrared spectroscopy spectra and the measurement of degradation efficiency of the main straw components further verify these microscaled observations.
Introduction Each year, million of tons of lignocellulosic wastes, consisting mainly of agricultural and industrial residues and municipal solid wastes, are produced worldwide (1). Their conversion to useful feedstuff or fuels using chemical or biological methods has been proposed and explored (2, 3). Anaerobic fermentation of lignocellulosic wastes is attractive due to environmental benefits, such as reducing the greenhouse effect, generating useful products, and increasing resource availability (4, 5). Various culture resources, such as anaerobic sludge, rumen fluid, and other sources of microorganisms, have been applied to degrade lignocellulosic wastes into volatile fatty acids and biogas (5–8). The rumen microorganisms have been found to be superior over other microbes for the degradation * Corresponding author. Fax: +86 551 3601592. E-mail: hqyu@ ustc.edu.cn. † School of Chemistry, University of Science & Technology of China. § Hefei University of Technology. ‡ National Laboratory of Micro-scale Material Structure, University of Science and Technology of China. 276
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of lignocellulosic wastes. The mixed rumen microorganisms, composed of bacteria, fungi, and protozoa, have all needed enzyme components with high enzyme activity for the degradation of lignocellulosic wastes. Also, the use of natural microbial consortia should be cost-effective. However, previous studies on the anaerobic fermentation of lignocellulosic wastes were focused on reactor configuration, operating parameters, microbial characterization, and process optimization (6–8). Little effort has been made to characterize the conversion processes on the microscale, for example, the changes in chemical topography of the substrates during bioconversion (3). Atomic force microscopy (AFM) is a powerful tool to get nanoscaled morphological information on a surface. The changes of morphological characteristics of the plant surface during biodegradation have been observed using scanning electron microscopy and transmission electron microscopy (9). However, preparation of the samples for examination damages the original surface morphology and structure. The changes on the surface can be observed directly by using AFM without sample preparation. AFM has made it possible to get high-resolution images of the interaction between bacteria and a solid surface (10, 11), cellulase action on cotton fibers (12), electrochemical corrosion of mild steel (13), and surface morphology of wood samples (14). The morphology of biomass could be observed directly with AFM, and structural characteristics could be analyzed topographically in the biotransformation of biomass. AFM images revealed that a compact layer of wax covered the outside of the straw, and that a network structure of cellulose and hemicellulose, with lignin localized on the surface of the network, was located at the boundary of the primary and secondary walls (15). The main objective of this study was to investigate the changes of morphology of lignocellulosic wastes, taking wheat straw as a representative substrate, during anaerobic degradation by using AFM and chemical analysis, and to further elucidate its biodegradation mechanisms. To the best of our knowledge, this is the first report of an investigation into the rumen fluid-mediated degradation of lignocellulosic wastes through the combinination of morphological observation by AFM with chemical analysis by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and gas chromatography/mass spectrometry (GC/ MS).
Materials and Methods Sample Preparation. Wheat straw was collected from a farm in the suburb of Hefei city, China. After being cleaned and dried, the straw was cut into stalks with a length of 1 cm. The fragments of the stem were dewaxed with toluene/ethanol (2:1, v/v) in a Soxhlet apparatus for 10 h. The dewaxed fraction was dried in a vacuum desiccator for subsequent use. Ruminal Inoculum and Incubation Conditions. The ruminal inoculums were obtained following the procedures described in the Support Information (SI). A total of 15 mL of the inoculum was dispensed into a Hungate tube under a CO2 atmosphere. Each tube contained three pieces of stem fractions. At the same time, 15 mL of the inoculum was autoclaved at 121 °C for 20 min and added into a Hungate tube as a control. In addition, another was added to 15 mL of Pfennig solution also as a control. Each control had four tubes, in which stem fractions came from the same straw source. The initial pH was set at 6.90. Tubes were placed in a water-heating incubator for agitation at 150 rpm and 39 ( 1 °C. Samples were taken at predetermined intervals, while the controls were sampled after 13 days. 10.1021/es071915h CCC: $40.75
2008 American Chemical Society
Published on Web 11/30/2007
FIGURE 1. Surface topography of the outside surface of straw after dewaxing. The fermented samples and the controls were rinsed with distilled water, resuspended in distilled water for 5 min in beakers, and then washed with distilled water again. After being cleaned, the samples were dried in a vacuum desiccator for analysis. AFM Observation. AFM observation was performed using a NanoScope IIIa Multimode scanning probe microscope (Digital Instruments Inc., Santa Barbara, CA). The images were scanned in the tapping mode in the air using an NP-S silicon nitride cantilever probe (DI, spring constant ca. 0.06 N m-1) with a resonance frequency of 250∼360 kHz. The scanning rate ranged from 0.5 to 3.0 Hz, and the scanning size varied from 1 to 10 µm. Both height and phase images were captured at the same time, and all images were measured at a resolution of 512 × 512 pixels. For each sample, many locations were measured, but only the representative AFM images are presented here. GC/MS Analysis. Three parallel experiments were performed to confirm the degradation of the lignin in the straw by the rumen microorganisms. Sample 1 was composed of 5 g of wheat straw, 1 g of cellulase, and 150 mL of acetic acid/sodium acetate buffer (pH ) 4.5). Sample 2 was composed of 5 g of straw and 150 mL of water, while samples 3 and 4 were composed of 5 g of straw and 150 mL of the diluted and sterile rumen fluids, respectively. Sample 1 was placed in a water-heating incubator for agitation at 150 rpm and 45 ( 1 °C. Samples 2 and 3 were incubated under similar conditions but at 39 ( 1 °C. After 5 days, the solution of each sample was filtered with a 0.45-µm micropore membrane and extracted with redistilled methyl tert-butyl ether. Each set of extracts was combined and dried with anhydrous magnesium sulfate and concentrated under reduced pressure. A GC/MS analysis was performed using a GC (Model 6890, Angilent Inc., U.S.A.) coupled to a GCT-MS apparatus (Micromass Ltd., UK) through an electron impact interface. Portions of 0.2 µL of concentrated extracts of the fermentation products were subjected to GC/MS determination. Chromatographic measurements were performed with a capillary column, DB-5 (30 m × 0.25 mm × 0.25 µm, Angilent Inc., U.S.A.). Ultrapure helium was used as the carrier gas at a flow rate of 1 mL min-1. XPS, FTIR, and Chemical Component Analysis. XPS measurements were conducted with an ESCALAB MK II electron spectrometer (VG Scientific Ltd., U.K.) using a monochromatd Al KR X-ray source at a base pressure below 5 × 10-8 Torr. Scanning was carried out over a wide binding energy range (0–1100 eV) at an electron takeoff angle of 90° from sample areas less than 1 mm in diameter. The relative amounts of differently bound carbons were determined from high-resolution C1s spectra. The overlapping peaks in the C1s region were resolved into their individual components
by using a curve-fitting program (XPSPEAK v4.0), a Tougaard background, and symmetric Gaussian curves. The main peak was assigned at 284.8 eV to aromatic and aliphatic carbon [C-(C, H), referred to as C1 in this work]. The chemical shifts relative to C1 used in the nonlinear least-squares curve fitting were 1.7 ( 0.1 eV for C-O (C2) and 3.4 ( 0.2 eV for O-C-O or C)O (C3). FTIR analysis was carried out using a Magna-IR 750 (Nicolet Instrument Co., U.S.A.). The dried straw sample was ground into a powder and then ground with KBr again and pressed at 40 MPa to form a uniform disk. Chemical components and their individual degradation efficiencies were measured with the methods described by Yue et al. (8).
Results Images of the Straw after Dewaxing. Typical AFM 3-D and phase images for the stem outside surface of wheat straw after dewaxing are shown in Figure 1. Part of the surface appeared to be covered by thin flakelets, which might be the residual plant wax, and aggregates of granules with sizes ranging from 20 to 130 nm, which were likely to be lignin (12). No exposed fibers were seen on the straw surface after dewaxing. Images of Straw Surface at Different Fermentation Times. The AFM images for the surface of the stem after 5 days of microbial degradation are similar to the ones for the dewaxing sample (Figures 1 and 2a). However, holes and cracks were observed on the outside of the stem and at their bottom, and exposed microfibrils could also be observed. These exposed fibers were short fibrils located in the outer layers with a diameter of 15–30 nm (arrow in Figure 2a). Since the amorphous materials covering the microfibrils were found to be hydrophobic in the AFM measurement (16), these structures preferably consisted of wax or lignin, rather than hydrophilic hemicelluloses. For AFM images of the surface of the stem after 9 days of anaerobic degradation, the majority of the surface consisted of nonfibrillar amorphous materials, as described previously. Typical structures of the exposed surface are shown in Figure 2b. Holes in the microfibril with a diameter of approximately 600 nm were observed, indicating that the microfibril was penetrated by the action of rumen microorganisms. On the stem surface, amorphous materials covered the microfibril. The granules covering the surface were found to be significantly reduced (Figure 2b). The blending of amorphous fibril and grains suggests that the lignin granules on the outer surface had been gradually degraded or resolved and that the amorphous materials might be the residual of hemicelluloses or a lignin-hemicellulose complex (14). In the inner layer, a bundle of fibril was oriented VOL. 42, NO. 1, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Surface topography of straw after anaerobic fermentation for (a) 5 days, (b) 9 days, and (c) 13 days. in the same direction, which was mainly composed of crystalline microfibrils. After 13 days of anaerobic fermentation with rumen microorganisms, the stem outer surface was still partially covered by granules. Simultaneously, microfibrils with a diameter of 20–60 nm occupied a larger area of the outer surface (Figure 2c). This was substantially different from the fibrils in the samples after 9 days of fermentation. These microfibrils were oriented in the same direction and ranged in layers. Presumably, such degradation occurred from outer to inner layers one layer at a time. Images of Straw Soaked in Sterile Rumen Fluid. Figure 3 illustrates the surface of the control sample soaked in the sterile rumen fluid for 13 days. The stem surface was fully covered by granules and flakelets. The control sample soaked 278
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in the Pfennig solution had similar surface characteristics (images not shown). A comparison among Figures 1-3 suggests that these granules and flakelets were very likely to be lignin and residual wax, respectively. Apparently, without the presence of action of rumen microorganisms, the lignin on the stem surface could not be dissolved in the buffer under the same conditions (Figures 1-3). GC/MS. To determine whether or not the lignin in straw was degraded by the rumen microorganisms, a GC/MS analysis was carried out on the extracts of the different straw samples treated by the action of rumen microorganisms. The results showed that 3,4-dimethoxyphenol and 3,4dimethoxybenzoic acid, two typical biodegradation products of lignin, were identified by their mass spectra in the extract of the straw samples treated by rumen microorganisms (mass
FIGURE 3. Surface topography of straw soaked in sterile rumen fluid.
FIGURE 4. Typical XPS wide-scan spectrum of straw soaked in Pfennig solution.
TABLE 1. O/C Atomic Ratios and Relative C1–C3 Peak Areas of Straw and Its Main Components
FIGURE 5. XPS spectra of C1s of straw soaked in (a) Pfennig solution, (b) sterile rumen fluid, and (c) rumen fluid.
relative area (%) material theoreticala lignin cellulose hemicelluloses wax measured wheat strawb wheat strawc wheat strawd
O/C
C1
C2
C3
0.33 0.83 0.80 0.04∼0.11 0.164 0.170 0.352
49 0 0 94∼100 81.9 81.7 65.7
49 83 83 0 13.5 12.9 25.5
2 17 17 0∼6 4.6 5.4 8.8
a Cited from Gustafsson et al. (16). b Wheat straw soaked in Pfennig solution for 13 days. c Wheat straw soaked in sterile rumen fluid for 13 days. d Wheat straw after 13-day fermentation.
spectra have been listed in the SI). However, no substances with the structural features of lignin decomposition products were detected in the other samples. XPS. The evolution of the O/C ratios and relative abundance of C1–C3 in the biodegradation could be used to characterize the changes of components on the straw surface (16, 17). A typical XPS scanning (0–1100 eV) spectrum for straw is shown in Figure 4. No surface elements other than oxygen (O) and carbon (C) were observed, except a small amount of nitrogen (N). The main components of the wheat straw surface had different theoretical O/C atomic ratios and relative C1–C3 peak areas, as shown in Table 1. Figure 5 illustrates the XPS spectra of C1s of the different straw samples. The peak-fitting results and O/C atomic ratios are summarized in Table 1.
FIGURE 6. FTIR spectra of straw soaked in (a) Pfennig solution, (b) sterile rumen fluid, and (c) rumen fluid. FTIR. The FTIR spectra of the different straw samples are illustrated in Figure 6. The corresponding assignments are listed in Table 2 (18). The relative absorbances of the characteristic bands of lignin at wavenumbers of 1459, 1432, 1323, 1252, and 1158 cm-1 decreased, whereas the relative absorbances of the characteristic bands of cellulose at wave numbers of 1378 and 1048 cm-1 slightly increased. This indicates a decrease in the relative fraction of lignin. These results further confirm that lignin was degraded by the action of rumen microorganisms. Degradation Efficiencies of the Main Components in Straw. The chemical components of straw after 13 days of VOL. 42, NO. 1, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Characteristic FTIR Bands of Straw material
wavenumber reported (cm-1)
wavenumber picked (cm-1)
assignment
cellulose
1666 1387 1049 1505 1460 1440 1325 1280 1155
1641 1378 1048 1515 1459 1432 1323 1252 1158
absorbed water bending O-H bending C-O-C pyranose ring skeletal vibrations aromatic skeletal vibrations aromatic methyl group vibrations aromatic skeletal vibrations syringyl ring breathing with CO stretching guaiacyl ring breathing with CO stretching aromatic CH in-plane deformation, guaiacyl type
lignin
TABLE 3. Degradation Efficiencies of Dry Weight, Lignin, Cellulose, and Hemicellulose after 13-Day Fermentationa
samples soaked in Pfennig solution samples soaked in sterile rumen fluid sample fermented in rumen fluid a
dry weight
lignin
cellulose
hemicellulose
18.4 ( 2.4% 18.2 ( 3.0% 40.8 ( 1.7%
7.8 ( 2.0% 7.6 ( 2.6% 25.5 ( 2.0%
14.8 ( 2.1% 13.5 ( 1.4% 44.0 ( 3.3%
22.4 ( 4.8% 24.0 ( 3.4% 43.2 ( 2.4%
Note: Standard deviations were calculated with three measurements.
incubation with rumen microorganisms and the two controls were also analyzed. The degradation efficiencies of dry weight, lignin, cellulose, and hemicellulose after 13 days of incubation are listed in Table 3. The sample treated by rumen microorganisms showed that all structural components were more highly degraded than the controls. Although lignin is a refractory component to microbial attack, comparing with cellulose and hemicellulose especially under anaerobic conditions, notable degradation efficiency was found for the samples treated by active rumen microorganisms, suggesting that lignin could be degraded or solubilized by the anaerobic rumen microorganisms.
Discussion Because of the recalcitrance of lignin, the low cellulolytic activity, and slow specific growth rates of the anaerobic microorganisms involved, the anaerobic conversion efficiencies of lignocellulosic wastes are usually very low in conventional bioreactors (2, 4). Our previous investigations and other studies have shown that the application of rumen microorganisms could enhance this process (6–8). In the present work, 25.5 ( 2.0% of the lignin in wheat straw was removed within 13 days of anaerobic fermentation by rumen microorganisms. Comparing with a 7.8% lignin removal in the control, the application of rumen microorganisms resulted in a 17.7% increase in lignin removal (Table 3). In a previous study on the fermentation of corn stover by rumen microorganisms, approximately 30% of the lignin was degraded within 10 days (6). The AFM images in the present work show the removal of lignin by rumen microorganisms (Figures 1 and 2). This further demonstrates the potential utilization of rumen microorganisms for the bioconversion of lignocellulosic wastes. However, the loss of lignin does not necessarily mean it is utilized by the rumen microorganisms, and it could also be attributed to its solubilization (19, 20). For instance, on the basis of 14C-lignocellulose test results, a previous study showed that most losses of lignin were attributed to solubilization from fiber (21). In our present work, the AFM images directly show the gradual remove of lignin granules from the external straw surface (Figure 2), while the GC/MS results indicate that the action of rumen microorganisms did decompose the lignin in straw, on the basis of a comparison among the samples with ruminal treatment and the control. However, a further investigation using molecular biological techniques is warranted to explore the dominated microbes 280
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in the rumen microorganisms, which are mainly responsible for the biodegradation of lignin in straw. Electron microscopy has been proven to be a useful tool in investigating structural changes of lignocellulose which is degraded by fungi and bacteria, and its utilization has significantly improved our understanding of the biodegradation of wood (21). Compared with electron microscopy, AFM is able to image surfaces at a spatial resolution without special sample preparation (22). In the present study, the AFM images after 5 days of fermentation clearly show the holes and cracks on the stem surfaces and suggest that tunneling might be one of the ways for rumen microorganisms to attack the stem. The 3-D images for the straw surface at different fermentation times in Figure 2 show that the surface traces changed significantly in the fermentation mediated by rumen microorganisms. The straw surface exposed to rumen fluid displayed nonuniform surface traces, indicating a gradual falling off of the granules and flakelets (Figure 2). Such a change could be attributed to the deterioration, degradation, or falling off of the surface materials because of the microbial attack. Phase images of samples are less commonly reported in the literature than height (topographic) images, because the different components that give rise to phase shifts of the cantilever are more complex. In spite of a limited ability to interpret the complicated phase images in a quantitative manner, phase images are useful because they provide a measure of sample heterogeneity (22, 23). The accuracy and reproducibility of tapping mode height profiles for many types of samples have been verified by a comparison of AFM images with other types of electron microscopy for gold nanoparticles, polysaccharides, and humic substances (24, 25). The phase images in Figure 2 clearly illustrate the change of the straw surface heterogeneity after anaerobic fermentation. This provides direct evidence to support the degradation of lignocellulose by rumen microorganisms. In addition to the morphological structure, the surface characteristics are also related to the variation of the molecular structure and corresponding local element distribution. In this case, XPS combined with solvent extraction has been used to evaluate the surface of lignin and extracted contents of the pulp (14). After anaerobic fermentation, the O/C ratios and relative abundance of C2 and C3 increased remarkably, while that of C1 decreased significantly (Table 1). Table 1 indicates that the lignin and wax on the stem surface were partially degraded by rumen microorganisms
and that the cellulose was exposed on the surface. This result is in good agreement with the AFM observations (Figure 2). The O/C ratio in the controls was lower, while the relative amount of C1 was higher than the theoretical values of lignin, suggesting that the stem surface still contained a large amount of wax. This is also confirmed by the AFM images (Figures 1 and 3). Anaerobic fermentation of lignocellulosic wastes is a very complex bioprocess; it is difficult to investigate such a process on the microscale level. In the present work, a combination of morphological observation by AFM with chemical analysis by XPS, FTIR, and GC/MS as well as component measurement was successfully used to explore the bioconversion process. Such a combination provides unique and useful information on straw biodegradation by rumen microorganisms. In the present work, a combination of AFM and XPS was applied to characterize the straw surface in anaerobic fermentation. AFM reveals surface morphology, while XPS gives chemical characteristics of surface regions. The combination of morphological and chemical information yields better insight into the characteristics of the straw surface. Such information could lead to a better understanding of the performance and mechanisms of the biodegradation of lignocellulose.
Acknowledgments The authors wish to thank the Natural Science Foundation of China (20377037 and 20577048), and National Basic Research Program of China (2004CB719703) for the partial financial support of this study.
Supporting Information Available Procedures to obtain inoculums and mass spectra of main fermentation products from straw are shown. This material is available free of charge via the Internet at http:// pubs.acs.org.
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