Langmuir 1993,9,1721-1726
1721
Associative and Colloidal Behavior of Lignin and Implications for Its Biodegradation in vitro Gianfranco Gilardif and Anthony E. G. Cass' Centre for Biotechnology, Imperial College of Science Technology and Medicine, London SW7 2AZ, U.K. Received November 2,1992. I n Final Form: April 23,1993 Lignin is a relatively complex aromaticbiopolymer that occursin woody plant cell walls. This biomaterial can be degraded by extracellularenzymes from white rot fungi,a process that has attracted much attention. Although the enzymes that catalyze such a biodegradation process have been identified and isolated, the rate of lignin biodegradation in vitro has been found to be quite low. The view presented in this paper is that this may be due to the way that the substrate, lignin, is presented to the enzyme. In fact, little is known about the substrate in ita natural physical state or about the modifications that it undergoes during the solubilization process. In this paper a solvent-extracted lignin from sprucehas been characterized in terms of ita molecular weight and size. Evidence is presented that shows that this lignin sample forms aggregates in a dioxanwater (51) solvent system, having a hydrodynamicradius of 60nm. Each aggregate is formed with an average of 256 molecules and has a cross-sectionalarea of 190nm2,as determined using a LangmuirBlodgett trough. Furthermore,in water solutionsthe pH was found to influence the molecular weight of lignin, as seen by high-pressure liquid chromatography and scanning electron microscopy. As the pH was lowered from 12 to 2, lignin aggregates with a discrete pattern of molecular weight incrementa. These resulta are interpreted in terms of electrostaticand van der Waals forces interactingbetween polar and apolar groupsof the ligninmolecules. These fiidings stressthe importanceof controlling the conditions under which lignin is presented to the ligninolytic enzymes during in vitro degradation experiments.
Introduction Lignin is one of the major components of the plant cell wall (20% by dry weight), along with cellulose and hemicelluloses. The aromatic nature of this biopolymer and ita extensive biodegradation by white rot fungi has attracted the attention of the biotechnologists. the exploitation of aromatic compounds derived from lignin breakdown is certainlyof interest of the paper and chemical industry. Early work suggested a view of lignin as a complex threedimensional polymer network; ita structure in wood was visualized as branched with linear chains cross-linked by a variety of interchain covalent bonds.' The strongest argument in favor of this "network theory" is that native lignin is insoluble in all neutral solventsand this, according to the theory, is due to strong covalent lignin-lignin bonds present in wood. The solubilization of this complex inevitably involves breaking of bonds, yielding molecular fragments, and leading to the polydispersity in molecular weight of soluble lignin derivatives. Some recent work has shed a new light on the behavior of lignin in solution, in respect of ita molecular weight; Dutta and Sarkanen2 reported size-exclusion chromatography data consistentwith an associativeprocess occurring among lignin molecules. These and previous resulta from the same groupw showed an increase in molecular weight of kraft lignin solutions over time. The authors suggested the presence of "noncovalent" interactions between individual lignin molecular components. ~~~
~
+ Current address ChemiatryDepartment,Leiden University,P.O.
Box 9602,2300RALeiden, The Netherlands. (1) Adler, E. Wood Sci. Technol. 1977,11, 169.
(2) Dutta,S.; Sarkanen, 5.In Materials Interactions Releuant to the Pulp, Paper and Wood Industries;Pasaretti,J. D.,Caufield,D.F.,Eda.; Materials Research Society: Pittsburgh, PA, 1990. (3) Gamer, T. M.; Iwen, M. L.; Sarkanen, S. Proceedings of the 5th Internutionul Symposium on Wood and Pulping Chemistry; TAF'PI Raleigh, NC, 1989; p 113. (4) Sarkanen, S.; Teller, D. C.; Abramowski, E.; McCarthy, J. L. Macromolecules 1982, 15, 1098. (5)Sarkaneq S.; Teller, D. C.; Stevens, C. R.; McCarthy, J. L. Macromolecules 1984,17,2588.
In an early work, Lindstroms showed an association process taking place between kraft lignin molecules, and speculated on the colloidal behavior of kraft lignin. This author also reported the presence of stable lignin sols in aqueous solution,whose extent of aggregation was followed by gel permeation chromatography and viscometric measuremente,however, no light scatteringmeasurementa were made. More recently, the colloidal dispersion of lignin in water has received further attention in view of biodegradation studies. Kurek and co-workers7 reported the preparation of colloidal dispersions of milled wood and enzymatically liberated lignin in water from dimethylformamidesolution,withno major alterations in molecular structure as judged by UV and IR spectra. The same authors claimed a higher ligninase activity, based on hydrogen peroxide consumption, toward the colloidal lignin compared with the ordinarily precipitated lignin.8 This finding shows that the physical state of lignin in solution may play an important role in the accessibility to enzymatic biodegradation in vitro. In a recent review, Lewis and Yamam0t.0~ discuee extensivelythe problem of lignin structure. Although this biopolymer is reported to have a random structure, many questions on this view remain open. Indeed, several data such as those concerning the ordered coordination in lignin biodegradation, as well as those on interaction with other cell wall components that are highly organized, indicate that is difficult to conceive a simply casual spatial distribution of lignin moieties. It may be more probable that lignin has a level of organization yet to be fully revealed. We recently presented NMR data that are consistent with the presence of domains of organization of the aromatic rings in the lignin network. A model involving (6)Lindstrom, T. Colloid Polym. Sci. 1979,257, 277. (7) Kurek, B.; Monties, B.; Odier, E. Holzforschung 1990,44,407. (8) Kurek, B.; Monties, B.; Odier, E. Enzyme Microb. Technol. 1990, 12. 771. (9) Lewis, N.G.; Yamamoto,E.Annu. Rev.Plant Physiol. Plant Mol. Biol. 1990,41, 455.
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0743-7463/93/2409-1721$04.00/0Q 1993 American Chemical Society
1722 Langmuir, Vol. 9, No. 7, 1993
stacking of aromatic rings is supported by computer simulated NMR spectra of lignin and molecular modeling of lignin fragments.10-l2 In this paper we report studies conducted on the associative and colloidal behavior of organosolv spruce lignin. This sample offers the advantage of being exposed to fairly mild solubilization conditions and, therefore, is structurally much closer to the native lignin than the kraft preparations studied previously. The molecular weight characterizationis followed by determination of molecular dimensions and studies of the light scattering properties of the lignin colloidal solutions. The associative behavior is studied as function of pH rather than time as in previous work.2
Materials and Methods Organosolv lignin samples were prepared from spruce wood chips, by hydrolysis in 50%aqueous ethanol and 75%aqueous ethylene glycol mixtures at 135 "C for 2 h, according to the procedure described by Sarkanen and co-worker~.~~ After the pulping reaction was stopped, the solvents were removed by evaporationunder reduced pressure,leading to the precipitation of the organosolv lignin. Molecular weights ( M W ) of organosolv spruce lignin were determinedby gel filtrationon a Varian 5OOO high-pressureliquid chromatograph(HPLC),with a Varian 2050variable wavelength detector, and a Spectra Physics SP 2050 integrator. A Zorbax HPLC column (Du Pont) with porous silica microspheres (PSM 60s) as a stationary phase and Nfl-dimethylformmide (DMF', Aldrich HPLC grade) as mobile phase were used. Solutions in DMF of 10 mg/mL polystyrene molecular weight standards (Du Pont) with MW of 800,2000,4000,9000,17 500, and 50 OOO, were used to calibrate the HPLC system. Organosolv spruce lignin was dissolved in DMF (0.25 mg/mL) and filtered througha 0.45pm pore sizepoly(tetrafluoroethy1ene) (PTFE)filter, and 10 pL was injected into the HPLC apparatus with a flow of 1 mL/min. Eluates were detected by measuring the absorbance at 280 nm, corresponding to the absorbance maximum of lignin. Lmgmuir-Blodgetttrough experimentswere performed on a home-built inshment, kindlymade availableby the Department of Chemical Engineering of Imperial College (London,U.K.). A lignin monolayer was obtained by deposition of 14 gL of a 1 mg/mL solution of lignin in chloroform-tetrahydrofuran (7:l) solvent mixture, on the water surface of the trough dish. After evaporation of the solvent mixture, the lignin monolayer was compressed by a movable barrier, while the surface pressure was measured by a movable float made with a filter paper soaked in chloroform and attached to a torsion wire. The experiments were performed at a constant temperature of 22 O C . Static light scattering experiments were carried out on organomlv lignin solutions in dioxane water (Sl), at concentrations ranging from 0.01 to 2.0 mg/mL, after filtering through a 0.45-m pore size PTFE filter. The intensity of the scattered lightwasmeasuredonaPerkin-Elmerluminescencespectrometer LS50, at 625 nm,a wavelength that was found to be unaffected
by the fluorescenceof lignin. The scattered light was read at a measurement angle of 90",in a square 1 cm cuvette. Dynamic light scattering experiments were performed in a Malven instrument on the same solutions that were used in the static experiments. A laser source at 632.8 nm was used, reading at a measurementangle of 90". The samples were positioned in a toluene bath (refractive index of 1.50), in a square quartz cell (1cm)at 25 "C. The particlehydrodynamicradiuswae calculated
(10) Gilardi, G.; Harvey, P. J.; Cass, A. E. G.;Palmer, J. M. In Biotechnology in Pulp and Paper Manufacture, Applications and EkndamentalInuestigations;Kirk, T. K., Chang H.,ma.,Butterworth: Boston, 1990; p 36. (I!) GiIardi, G. SpectroscopicStudiesof Lignin BiodegradationPh.D. Thesr, Univereity of London, 1991. (12) G h d i , G.; Caw,A. E. G.Manuscript in preparation. (131 Sarkanen. S.:Teller. D. C.:. Hall. . J.:. McCarthv, J. L. Macromolecu.les. 1981,14, 426:
Gilardi and Case with a Malven Correlator system, assuming a spherical shape and a sample viscosity of 1.167 cP. Nuclear magneticresonance(NMR) experimentswere carried out on a Bruker AC200 NMR spectrometer operating at 200 M H z for the hydrogen nuclei. Spin-lattice proton relaxation times of lignin were measured in deuterateddioxane-water (61) solvent mixture (100 mg/mL) in 5 mm NMR tubes (Gold Label, Aldrich). A spectralwindow of 2403 Hz was used to collect 4096 data points zero filled to 8192 data pointe, a 4 2 pulse of 7.6 ma was used and 16 transients were averaged. The spin-lattice relaxationtimes were determinedwith an inversionrecovery pulse sequence (r~-rr/2-collect).A recovery delay of 10 s was used between each experiment, and a series of interpulse delays was used ranging from 0.1 ms to 10 s. The relaxation values were calculated using the Bruker DISR89 software facilities on an Aapect 3O00 computer. Chemical shifte were referred to sodium 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionate. For the experiments on the effect of the pH on the MW of lignin,the organoeolv spruce lignin was solubilized in NaOH 0.1 M, at a concentration of 50 mg/mL aqueous solution, to give a pH of 12. The solution was divided into"a, whose pH was corrected by addition of 0.1 M HCl to reach pH 8, pH 6, pH 4, and pH 2. The final volume of each solution wae adjusted by addition of distilled and deionized water to give a final lignin concentrationof 1 % (w/v). The lignin solutionsand suspensions so obtained were divided into two pools: one was treated for
HPLC gel-filtration molecular weight analysis, the other was analyzed by a "ingelectron microscope (SEM). HPLC gel-fdtration: Lignin suspensionsat differentpHs were freeze-dried, and the powder resuspended in NJV'-dimethylformamide in the following concentrations: pH 12,l.O mg/& pH 8, pH 6, and pH 4,0.40mg/& pH 2,0.50 mg/ml; untreated control, 0.25 mg/mL. The HPLC gel-filtration analysis was conducted under the same conditions previously described. SEM: each lignin suspension was well shaken, and one drop was spotted on a glass elide (10mm in diameter). The specimens were rapidly immersed in Acton 22 slueh, cooled by a liquid nitrogen bath. After the quick freezing step, the aamples were transferred in a freeze-drier on a cold plate maintained at -30 "C. The dry specimenswere then m t e d with gold and examined on a Phillips 500 scanning electron microscope.
hulta Determination of the Average Molecular Area of Lignin. A relatively simple instrument able to estimate molecular dimensions is the Langmuir-Bladgett trough. This instrument is designed to measure the surface pressure a of a molecular monolayer floating on a nonmiscible solvent, while varying the area available to the monolayer itself. The plot of r against the area available to the molecule is an isotherm that shows the behavior of r with the variation of the physical state of the monolayer. In a typical isotherm, the region of steep variation of a with the area corresponds to the liquid condensed stage,where the molecules occupy an area equal to their molecular size. By extrapolating the slope of r to intersect the x axis (r= 0),the area value on this point is the 'limiting area", that can be considered very close to the cross-sectional area of the m o l e c u l e ~ . ~ ~ J ~ In this study this technique was applied to organosolv lignin to calculate the average cross-sectional area of the molecule, and the isotherm obtained is shown in Figure 1. The isotherm of Figure 1 shows the typical pattern of a monolayer of molecular species as a function of decreasing area available. The first part of the curve (G)exhibita surface pressure values lees than 0.36 mN m-l, which is indicative of high area available associated with a high dilution of the molecules with gaslike behavior. This is followed by a step (LE)in which a increases to 6.3-8.0mN (14) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley-Intemience: New York, 1966. (16) Mohwald, H.A. Annu. Reo. Phys. Chem. ISSO, 41,441.
Biodegradation of Lignin
Langmuir, Vol. 9, No. 7, 1993 1723
a35-
aael-
LC
II
I 200 - I
l
I
/
0.0
0.2
0.4
0.6 0.8 1.0 ILigninYmq m ~ '
1.2
1.4
Figure 2. Intensity of light scattered by organoeolv spruce lignin solutions at 625 nm,as a function of lignin concentration. x 10 nm2
Figure 1. Surface pressure versus area per molecule isotherm obtained on the LangmwBlodgett trough for a organosolv spruce lignin monolayer spread on a water surface. m-l and is indicative of liquidlike character of the monolayer, due to a decrease in the area available for each molecule. The liquid-expanded state (LE) is followed by an intermediate state (I) in which the area occupied per molecule approaches the molecular size. When this point is reached, a steep increase in the slope is observed (LC), and the system appears to be in a liquid condensed state, with surface pressure up to 45.5 mN m-l. A further decrease in area causes destruction of the monolayer, with drop in surface pressure values as observed in (SI. The calibration of the area available for each molecule critically depends upon the molecular weight of the sample under investigation. Although ligninis a complex polymer with a degree of polydispersity in dioxane/water solutions (see the discussion of the light scattering results later in this paper) HPLC gel-filtration measurements in DMF suggest an average molecular weight of 70000 and a relatively narrow peak width. These measurements were made on a column calibrated with molecular weight standards up to 50 OOO as described in the materials and methods; the estimate of 70 OOO is based upon a linear extrapolation of the log(mo1ecular weight) vs retention time data for these standards. Figure 1 showsthat the steep increase in surface pressure corresponding to the liquid condensed state of the lignin monolayer occurs for an area per molecule of 190nm2.As this is the stage a t which each molecule occupies its molecular area, 190 nm2can be taken as an average value for the cross-sectionalarea of the ligninmolecules analyzed. Given the complex structure of lignin, it is difficult to predict the molecular arrangement at the water-air interface; however, in the light of molecular area and molecular weight it is possible to make some suggestions. It is reasonable to assume that the lignin monolayer is spread on the water surfacesuch that hydrophilic moieties, generally represented by polar groups of the side chains, that are in close contact with the water interface, whereas apolar aromatic moieties may be exposed to the air phase. A similar model has been reported in literature for trip-cresyl phosphate molecule^,^^ showing a cross-sectional molecular area of 0.95 nm2. It can be calculated that a lignin molecule of average MW 70 OOO may contain 380 C-9 aromatic units of average M W 183 g/mol. Extrapolating from the values reported in literature for the tri-
p-cresyl phosphate molecule, a lignin bearing 380 aromatic units could be expected to form a monolayer with an approximate area of 120 nm2. The higher value of 190 nm2experimentally obtained suggests that indeed lignin forms a monolayer on the water surface, and the higher value is accounted for by the C-3 side chains and methoxyl groups attached to aromatic units, that contribute to a more expanded structure than that of tri-p-cresyl phosphate molecule. Aggregation of Lignin in Solution: Light Scattering Evidence. Light scattering measurements are based on the light scattered from translational and rotational degrees of freedom of particles. In fact, when light impinges on matter, the electric field of the light induces an oscillatingpolarization of the eledrons in the molecules. These behave as secondary sources of light and subsequently radiate or 'scatter" light, a phenomenon commonly called Rayleigh scattering. Frequency shifta due to loes of energy by photons, the angular distribution, the polarization, and the intensity of the scattered light are determined by the size, shape, and molecular interactions in the scattering materials. In this research, organosolv spruce lignin samples were studied by static and dynamic light scattering techniques to investigate the potential aggregation phenomenon of lignin in solution. As a preliminary experiment, the intensity of the light scattered by series of solutions of lignin at different concentration in dioxanewater (5:l) was measured. The results obtained from this experiment are reported in Figure 2. From Figure 2 it can be observedthat a dramatic increase in scattered light intensity is found between 0.5 and 0.8 mg/mL lignin concentration. Because lignin solutions exhibit strong absorption in the W region with a tail in the visible region, it was not possible to extend light scattering measurements to concentrations above 1.5 mg/ mL, due to interference from the intense absorption. The step increment in scattered light intensity above 0.5 mg/ mL suggests that an aggregation process is occurring. As the scattering tends to a plateau above 0.8 mg/mL, this may be an indication of formation of discrete particles, whoee assembly requires more than one molecule of lignin. The reversible self-asaemblingbehavior occurs at concentration above 0.5 mg/mL, which would correspond to the so-called critical micellar concentration in amphiphile solutions. The apparent increase in scattered light between zero and 0.5 mg/mL may be a consequence of multiple scattering at these concentrations.
Gilardi and Cas8
1724 Langmuir, Vol. 9, No. 7, 1993
Table I. 2'1 Relaxation Times of Dioxane-Water 6 1 with and without Lignin and at Different Temperature#
1.
Tils 200 MHz 25 OC without with lignin lignin 6.57 1.75 (0.011) (0.023)
... .
-0.4
4
.
*
0
20
40
60
*..,;.,.
wn i
chemical 300 MHZ With shift,ppm 25OC 70 O C 4.02(at 25 OC) 2.10 3.23 3.51 (at 70 "C) (0.017) (0.032) water 3.58 4.94 3.32 5.29 >10 dioxane (0.018) (0.020) (0.007) a Data are reported in seconds, and the standard deviations are indicated in parentheses.
,
80
Time/ps
Figure 3. Semilogarithmic plot of the autocorrelation function against time in dynamic light scattering by a solution of lignin in dioxanelwater at a concentration of 0.8 mg/mL. Data points are shown as black dots and the solid line is the fit to a four-term polynomial. The insert shows the residuals for the fitted line.
To further investigate this phenomenon, the same lignin solutions were examined by dynamic light scattering, and the results are reported in Figure 3. The nonlinear character of the curve in Figure 3 is consistent with a polydisperse sample, and under these conditions it is difficult to obtain reliable values for the diffusion coefficient and hence the hydrodynamics radius of the scattering centers. One approach is cummulant analysis16 where the data are fitted to a polynomial in (-d2where The solid line in T is the time (discussed by Pecora"). Figure 3 shows the results of a four-term polynomial fit to the individual datum points shown as dots. It is clear from the insert to Figure 3 that this is not a completely satisfactory fit as regards the distribution of residuals, but given the difficulties of reliably interpreting terms of higher order than (-rI2 it is considered adequate for our purposes. In this type of analysis the coefficient of the (-7) term is dependent on the average translational diffusion coefficient while the coefficient of the (-+ term is a measure of ita deviance. The normalized value of this latter term is often referred to as the "quality factor" and values of less than 0.02 are indicative of a clean monodisperse sample. Our results yield a quality factor of 0.9 showing that under the conditions of the experiment the samples are indeed polydisperse. If we calculate the average diffusion coefficient and assume a spherical shape, then using a sample viscosity of 1.17 cP, these aggregates are found to have a hydrodynamic radius of 60 nm, with no significant variation between 0.8 and 2.0 mg/mL lignin concentrations. Experimenta carried out on samples with lower lignin concentrations did not show defined decay pattern, and samples with higher lignin concentrations presented absorption interference. The assumption of a spherical shape for the aggregates is supported by early viscosity measurements (Sarkanen and Ludwig1*) that reported lignin solution viscosity values much lower than those measured for linear (cellulose) and branched (xylan) cell wall polymers of equal molecular weight. From the average hydrodynamic radius value, it can be calculatedthat the averagesurface of the sphericalparticles (16) Koppel, D. E. J. Phys. Chem. 1972,57,4814. (17) Pecora,R. Dynamic Light Scattering;PlenumPress: New York, 1985. (18) Sarkanen,K.V.; Ludwig,C . H.Lignins: Occurrence,Formation, Structure and Reactions.; Wiley-Interscience: New York, 1971.
is 480 X lo2 nm2. Given the average molecular croessectional area value of 190 nm2 calculated with the Langmuir-Blodgett technique, each aggregate has an average assembly of 256 lignin molecules. It may be interesting at this point to consider two examples of well-studied colloidalsystems: latex particles and soap micelles. The former are heavily cross-linked polymer particles, which behave as rigid balle; the letter are formed by amphiphfic molecules such as long chain fatty acids or phospholipids, that in water tend to gather into spherical aggregates (micelles), in which the polar heads face outward to the polar water molecules, and the hydrophobic chains lie buried in the aggregate's core.l8 Given the absence of obvious polar and apolar heads in lignin moieties, it seems difficult to explain the aggregation process in terms of micelles. Considering the complex structure of lignin molecules, it is difficult to predict to predict their arrangement in the aggregates, but it may be possible however to identify apolar patches formed by aromatic rings and polar sites along the side chains: these patches are likely to combine at intra and intermolecular level leading to the aggregates. Influence of Lignin on Proton NMR Relaxation Times of the Solvent. The effect of the presence of lignin aggregates on the solvent molecules was studied by measuring the spin-lattice proton NMR relaxation times (2'1) of the solvent resonances. This parameter is sensitive to molecular motions, and ita variation can be diagnoetic of increased or restricted molecular mobility. Table I showsthe TIvariations of the solvent resonances, dioxane-water (5:1),without and with the addition of lignin, and at different temperatures with lignin. From these data it can be observed that the addition of lignin in the solvent system causes a dramatic decrease in the water relaxation time (73%), while a considerably smaller effect is observed on the dioxane TIvalues (33% 1. Furthermore, increasing the temperature causes a marked increaseinthedioxanerelaxationtimes(>100% variation), but just a limited effect is observed on the water peak (35%variation); the water resonance also shifts to higher fields, from 4.02 to 3.61 ppm. These data suggest a restriction in water mobility due to the presence of lignin; this may be indicative of the presence of hydrogen bonds between the water molecules and the polar side-chaingroups of the polymer. Thie may be interpreted with a "trapping" of the water molecules in the lignin aggregates. Effect of pH on Lignin Molecular Weight: HPLC Gel Filtration and SEM. These experiments are concerned with the study of the influence of pH on the lignin aggregation process. While the HPLC gel-filtration molecular weight determinations were carried out on lignin (19) Prost, J.; Rondelez, F. Nature 1991, Suppl. to uol. 360, 11.
Langmuir, Vol. 9, No. 7,1993 1725
Biodegradation of Lignin
"
20
40
60
80
12 0
PH
Figure 4. Results from molecular weight analysis of organosolv lignin exposed at different pHs (valuesare indicated in the graph).
samples that were resuspended in NJV'-dimethylformamide (DMF) after pH treatment, the scanning electron microscopy experiments were carried out on freeze-dried specimens obtained from a drop of lignin suspension a t a certain pH. In both situations the final observations are believed to reflect the modifications in the molecules while in solution, during the pH variation. The HPLC gel-filtration MW determinations were carried out on organosolv spruce lignin samples that were exposed to different pHs ranging from 10 to 2, freezedried, and resolubilized in DMF. The molecular weight analysis is reported in Figure 4. The molecular weight analysis shows a significant decrease in the low MW components from 48% at pH 12 to 19% at pH 2, accompanied by an increase in high MW components from 18% a t pH 12 to 65% at pH 2. Interestingly the HPLC chromatograms showed that the variations on the peak features, induced by the sample pretreatment a t various pHs, are not continuous but discrete: the decrease in the area of certain peaks is not due to a general broadening caused by more polydispersity, but an increase in the area of defined peaks is observed. These data suggest that a pH-dependent aggregation process occurs; moieties a t low molecular weight seem to assemble into high molecular weight aggregates. The pH dependence of the MW variations may indicate that polar interactions between lignin's ionizable groups, such as carboxylic and phenolic, are involved, and although DMF can well solubilize these aggregates, it has no influence on these interactions, possibly due to the absence of ionizable groups in DMF molecules. The same lignin samples were freeze-dried on microscope slides, and observed with a SEM. The effect of pH on lignin is shown in Figure 5. The pictures show a gradual transition from a very fine layer of grain particles at pH 12,to a spongelike arrangement a t intermediate pHs, and a more fibrous organization a t low pH. The SEM observations have a parallel in the literature, where humic and fulvic acids have also been reported to behave in a similar manner under the same conditions.20 These molecules have a polyaromaticstructure, that very closely resembles the chemical composition of lignin. This suggests an aggregation between lignin units that becomes evident when the pH is lowered to the acidic region, as lignin passes from a soluble to a colloidal aggregated form. According to the classical description of molecule aggregates, there are two principal forces involved in such process: the electrostatic and the van der Waals forces.lg The van der Waals interaction is always present, and it is due to the difference in polarizability between the molecules and the solvent. When the molecules bear ionizable groups, there is also an electrostatic repulsion, which is screened by the counterions present in solution. (20) Chen, Y.;Schnitzer, M.Soil Sci. SOC.Am. J. 1976,40, 682.
Figure 5. SEM micrographs of organosolv spruce lignin exposed to pH 12 (A, X640) and pH 2 (B, X640).
-XH H
H
a
a
Region of hydrophilic interactions
Region of hydrophobic interactions
Figure 6. Schematic representation of possible interactions between two lignin molecular fragments in a lignin aggregate.
As lignin contains such groups, the combination of these forces may be sufficient to describe its colloidal behavior and tendency to aggregate. It may be inferred that at high pH the lignin particles contain carboxyl, phenolic, and hydroxyl groups dissociated and therefore are negatively charged. Under these circumstances, the electrostatic repulsion between molecules is large enough to stabilize the solution against aggregation. As the pH is lowered, the electrostatic repulsion becomes weaker, the van der Waals and the hydrogen bond interaction become dominant, and a progressive aggregation is possible. This phenomenon may not be due to the simple
Gilardi and Cuss
1726 Langmuir, Vol. 9, No. 7,1993 precipitation rate of lignin a~ a function of pH, as the shape and feature of the polymer changes significantly, and the MW determination by HPLC gel-filtration has shown as this situation persists when lignin is completely resolubilized in organic solvents. Moreover, MW variations have been found to be discrete rather than continuous. Although the aggregation process ie reversible in water, organic solvents such as DMF have no effect, probably due to an inability to change the electrostatic interactions between the lignin aggregates.
important role in biodegradation studies carried out in vitro with purified enzymes. The physical state of lignin in solution could have a substantial influence on its accessibility to the enzymatic attack. This may offer an explanation to the early lack of success reported on degradation studies carried out in vitro with purified ligninase,21while recent studies by Kurek and co-workers* and Hammel and Moenn have found lignin degradation by carrying the experiments in solutions containing 1020% of organic solvent (DMF).
Conclusions The main conclusion to be drawn from the results
Acknowledgment. We gratefully acknowledge Dr. P. J. Harvey and Professor J. M. Palmer (Biology Depart-
obtained from different techniques reported in this paper is that indeed the lignin sample studied forms aggregates under the conditions analyzed. These aggregates do not however continue to grow in size until precipitation occurs and this probably reflects a dynamic equilibrium that is a consequence of both the structure of the lignin molecules and the solvent. In these studies dioxane is the major component of the solvent (6 parts dioxane to 1part water) and the driving force for aggregation may be to achieve maximum solvation of the polar side chains by water molecules while maintaining favorable interactions of the nonpolar parts of the molecule. This phenomenon may be relevant as it can affect the molecular weight characterization and may also play an
ment, Imperial College, London) for the many useful discussionsand comments, and for making availableHPLC and SEM facilities. We also thank the Department of Chemical Engineering (Imperial College, London) for making available light scattering and Langmuir trough facilities, in particular P. Hartley for the useful comments on the Langmuir trough experiments. G. Gilardi acknowledges with gratitude Montedison S.p.A. (Italy) for financial support. ~~~~
~
~~
(21) Haemmerli, S. D.; Leisola, M.S.A.;Fiechter,A. FEMSLett. 1986, 36,33. (22) Hammel, K. H.; Moan, M. A. EnzymeMicrob. Techml. 1991,13, 15.
