- Email: [email protected]
Lignin-depolymerizing activity of Streptomyces
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LIGNIN DEPOLYMERIZATION
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strated by ESR spectroscopy. Kersten et al. 9 detected the cation radicals of methoxybenzenes by ESR spectroscopy; Hammel et al. ~o detected radicals from dimeric model compounds oflignin through ESR spin-trapping techniques. Largely through the results of these two studies, a generalized mechanism for lignin degradation can be formulated. This generalized mechanism involves a central role for substrate aryl cation free radicals. Cation radicals can undergo a wide range of reactions; the type of reactions can be affected by the ring substituents. Substrates with a-hydroxy-containing propyl side chains (prominent in lignin) preferentially undergo carbon-carbon bond cleavage.t° Methoxybenzenes cation radicals tend to hydrate and demethylate for form formaldehyde and benzoquinones.9 Because the cation radicals are stable enough to diffuse away from the active site into the "bulk phase," their fate can also be dependent on the components of the bulk phase. Thus the pH, concentration of dioxygen, and concentration of other radicals can affect the addition of H20, addition of dioxygen, and dimerization with other radicals (reactions all observed with lignin models). Much of our understanding of the chemistry of cation radicals has been provided by Snook and Hamilton. 21 These workers studied the formation and degradation of aryl cation radicals in chemical systems. These studies have provided a model for ligninase catalysis; they have indicated that mechanistically, the chemistry of cation radicals accounts for most if not all of the prominent reactions observed in lignin biodegradation. Carboncarbon bond cleavage of propyl side chains, loss of methoxyls, oxidation of benzylic hydroxyls, and ring opening are mechanically consistent with a free radical mechanism. It is thus apparent that the future utilization of ligninase will require not only an understanding of ligninase catalysis, but also the chemistry of cation radicals. 21M. E. Snookand G. A. Hamilton,J. Am. Chem. Soc. 96, 860 (1974).
[24] L i g n i n - D e p o l y m e r i z i n g
Activity of Streptomyces
B y DON L. CRAWFORDand ANTHONY L. POMETTO III
Lignin is a complex phenylpropane polymer consisting of coumaryl, guaiacyl, and syringyl moieties linked together by numerous linkages, but primarily by the fl-aryl ether bond) Biodegradation of this recalcitrant R. L. Crawford, "Lignin Biodegradationand Transformation,"Wiley(Interscience),New
York, 1981. METHODS IN ENZYMOLOGY, VOL. 161
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form r--,;~erved.
250
LIGNIN
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polymer by Streptomyces is by an oxidative process in which the ether bonds are cleaved and the phenylpropane side chains are modified or removed.2 Some low-molecular-weight aromatic intermediates are released from lignin as a result of these reactions, but once produced they may or may not be catabolized by specific Streptomyees. 2 A principal result of fl-aryl ether bond cleavage is the substantial depolymerization of lignin. While Streptomyces-mediated depolymerization results in the production of relatively small amounts of single-ring aromatic intermediates, it results in the release of large amounts of a microbially modified water-soluble lignin polymer that is precipitable from aqueous solutions when they are acidified. This polymer has been named acid-precipitable polymeric lignin (APPL). 3 APPLs are the principal initial product of lignin degradation by the ligninolytic Streptomyces that have been studied thus far? In this chapter, we describe a simple spectrophotometric assay for monitoring Streptomyces-mediated solubilization of lignin when cultures are degrading lignocellulosic substrates in submerged cultures or in solid-state fermentations. We also describe a simplified lignin acidolysis procedure which is important for chemically characterizing APPLs in order to establish their lignin origin, to confirm that fl-aryl ether bonds in the original lignin were cleaved, and to demonstrate that lignin depolymerization occurred. Both the spectrophotometric and chemical assays are valuable for use in screening for new lignocellulose-decomposing microorganisms having the ability to depolymerize and simultaneously solubilize lignin. Cultivation of Streptomyces on Lignocellulose The preparation of the lignocellulose substrate and of log phase cells to be used as inoculum in solid-state fermentation and submerged culture systems is described in Chapter [5] in this volume. In this discussion, solid-state fermentations are described for Streptomyces viridosporus T7A (ATCC No. 39115) and a submerged culture system is described for Streptomyces badius 252 (ATCC No. 39117). These Streptomyces are examples of the two known types of lignin-depolymerizing actinomycetes,4 either of which might be encountered when using the lignin-depolymerization assay.
Solid-State Fermentation By following the procedures outlined in Chapter [5], 5 ml of late log phase cells of S. riridosporus is inoculated into 0.5 g of sterile ground and 2 D. L. Crawford and R. L. Crawford, Enzyme Microb. Technol. 2, 11 (1980). 3 D. L. Crawford, A. L. Pometto III, and R. L. Crawford, Appl. Environ. Microbiol. 45, 898 (1983). 4 j. R. Borgraeyer and D. L. Crawford, Appl. Environ. Micrbiol. 49, 273 (1985).
[24]
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extracted corn (Zea mays) lignocellulose, and incubations are carried out in cotton-plugged 250-m1 flasks. Incubation is at 37 ° in a humid incubator. Sufficient replicates are incubated so that at least three flasks can be harvested at each time interval to be examined, and a similar number of controls containing sterile lignocellulose moistened with only sterile meditim are incubated as well. The number of samplings per experiment will vary with the metabolic rate of the organism being studied; however, in initial experiments flasks should probably be harvested in replicates of three at 4-day intervals. After inoculation, time zero inoculated and control flasks are retained for immediate assay, and the remaining flasks are placed into the incubator. To prevent moisture loss flasks can be placed in a plastic bag along with a beaker of water to ensure that a humid environment is maintained. At periodic time intervals, three inoculated and three control flasks are removed for assay. Each flask is assayed for its content of polymeric water-soluble lignin (APPL), using the turbidometric procedure described below. Results are reported as averages of three replicate values with standard deviations.
Submerged Culture System Following the procedures described in Chapter [5] in this volume, 50 ml of late log phase cells of S. badius is inoculated into 5 g of sterile ground and extracted corn lignocellulose in a 2-liter flask, and the culture is preincubated as a solid-state fermentation at 37 ° in a humid incubator for 3 to 4 days. At the end of this period, 1.0 liter of additional liquid medium [0.6% (w/v) yeast extract in mineral salts] is added. This medium consists of 6.0 g of yeast extract (Difco Laboratories, Detroit, MI), 5.3 g Na2HPO4, 1.98 g KH2PO4, 0.2 g MgSO4"7H20, 0.2 g NaC1, 0.05 g CaC12" 2H20, plus 1.0 ml of trace element solution (6.4 g CuSO4" 5H20, 1.1 g FeSO4"7H20, 7.9g MnC12"4H20, 1.5g ZnSO4"7H20/liter of water) 5 per liter of deionized H20, adjusted to pH 7.1 - 7.2. After addition of the liquid medium, the culture is shifted to shaking incubation at 100 rpm and 37 °, and cultures are incubated in replicates of three. Three uninoculated controls prepared using only sterile medium in place of cell suspension are also incubated, and these controls are carded through the same sequence of manipulations as were the inoculated cultures. A zero time 10-ml sample of culture medium is taken aseptically at the time that the liter of medium is added to each of the solid-state fermentation flasks. Additional 10-ml samples are taken at 2-day intervals. Each sample is then assayed for soluble lignin (APPL) by the turbidometric procedure described below, and results are reported as averages of three replicate values with standard deviations. 5 T. G. Pridham and G. Gottlieb, J. Bacteriol. 56, 107 (1948).
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Turbidometric Assay for Water-Soluble Polymeric Lignin F r a g m e n t s
Solid-State Fermentation To each solid-state fermentation flask to be assayed, 125 ml of distilled water is added. The flask is then placed in a boiling water bath or steamer for 1 hr, during which time it is shaken periodically. Next, the suspension is filtered through preweighed filter paper (Whatman No. 54) to recover the residual lignocellulose/biomass for substrate weight loss determinations and later chemical analysis of the lignocellulosic residue (see below). For spectrophotometric assay of APPL, a 2.0-ml portion of the filtrate is pipetted into a cuvette, 0.1 ml of concentrated HCI is added to acidify the sample, and the solution is thoroughly mixed. This acidified sample is allowed to stand at least 1 hr at 4°, over which time any APPL or other acid-precipitable material present will precipitate. The sample is then remixed, and its optical density (OD) is determined at 600 nm against a water blank. Several repeat readings are taken to minimize variation of data due to the settling of the precipitate during measurement. An average OD reading is then calculated. When a reading is taken, it is also advisable to calculate average values based upon three to four separate 2.0-ml sampies. By periodically harvesting flasks and performing turbidometric assays as described, a turbidity versus time plot can be drawn, and production of APPL by the culture over time can be monitored.
Submerged Culture System The assay for lignin solubilization by cells growing as submerged cultures is simpler than for those growing as solid-state fermentations since the cultures do not have to be subjected to the hot water extraction procedure. Ten-milliliter samples of culture supernatant are aseptically withdrawn from the incubating culture at 2-day intervals, and they are filtered to remove any insoluble material. The filtrate is then assayed for solubilized lignin turbidometrically at 600 nm as above.
Correctionfor Protein in the Precipitates The only disadvantage of relying solely on the turbidometric assay for following APPL production by Streptomyces is that extracellular proteins may also precipitate when samples are acidified, and thus inflate the APPL values. 6 This problem seems to be more evident in the submerged culture 6 A. L. Pometto III and D. L. Crawford, Appl. Environ. Microbiol. 51, 171 (1986).
[24]
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system than in the solid-state fermentation system. It is relatively easy to correct for protein contamination by assaying air-dried APPL precipitates for crude protein content by the Kjeldahl procedure (see below and Chapter [5] in this volume). Confirmation of Lignin-Solubilizing Activity If a specific microbial strain grows well on lignocellulose in solid-state or submerged culture and produces increasing amounts of APPL-like material over time as shown by the turbidometric assay, then it can be considered tentatively as a lignin-depolymerizing microorganism. The acid-precipitable material measured by the turbidometric assay must, however, be examined to confirm that it is lignin in origin and that a correlation exists such that the appearance of solubilized lignin corresponds to a similar pattern of loss of insoluble lignin from the lignocellulose substrate (see Chapter [5] in this volume). 4
Confirmation for Solid-State Fermentations It is desirable first to quantify the exact amount of APPL present in filtrates taken from the different-aged cultures discussed above. When each flask is harvested, the total volume of filtrate is determined, and then the filtrate is acidified to pH 1 - 2 with concentrated HC1. The precipitate that forms is collected by centrifugation (16,000 g) in preweighed centrifuge bottles. The supernatants are discarded. Each precipitate is washed once with acidic water, and then it is air dried at 50 ° for 24 to 48 hr. The weight of the total recovered APPL is next determined to ___0.1 mg accuracy, and with the total volume removed for turbidometric assay taken into consideration, the total milligram weight of APPL recovered from the culture is calculated. After the dry weight of APPL is determined, the Klason lignin, carbohydrate, protein, and ash contents of the APPL are determined (see Chapter [5]). From these data it is possible to correlate the actual weight of lignin-derived APPL with the OD60o readings, and thereby generate a standard APPL curve for the organism under study. The standard curve is presented as a graph of OD60o against mg/ml APPL values. One should also calculate the percentage weight loss of lignocellulosic substrate at each time interval by weighing the air-dried residues recovered at each filtration step. The crude protein, lignin, and carbohydrate contents of the insoluble residues can also be determined so that cell mass increases and lignin and carbohydrate depletion from the lignocellulosic substrate can be calculated and compared with the rate and extent of APPL production by specific ligninolytic Streptomyces (see Chapter [5] in this volume).
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Submerged Culture System Once the time course of apparent lignin solubilization has been determined, tentatively identified lignin-solubilizing cultures are again grown on lignocellulose as described above. However, this time the cultures should be grown in 250-ml flasks containing 0.5 g of lignocellulose, and three replicate flasks should be harvested at each time interval. Sterile uninoculated controls should be incubated in the same fashion. Preincubation as solid-state fermentations is used as before, which in this case requires an inoculum of 5 ml of late log cells (for S. badius), followed by 3 - 4 days of preincubation at 37 °. Then 100 ml of the 0.6% (w/v) yeast extract- mineral salts medium is added prior to aerobic shaking incubation at 100 rpm and 37 °. At the time of initiating the submerged culture portion of the incubation and at 2- to 3-day intervals thereafter, three flasks each from the inoculated cultures and the uninoculated sterile controls are harvested. The flasks are placed in a boiling water bath or steamer for 1 hr, and the residual lignocelluloses are recovered by filtration through preweighed filter paper. The residues are air dried and weighed as described above so that the rate of lignocellulose weight loss over time can be calculated. Each residue is also chemically analyzed for its crude protein, lignin, and carbohydrate contents (see Chapter [5] in this volume) so that increases in actinomycete cell mass and lignin and carbohydrate depletion rates can be calculated. The filtrates are transferred into preweighed centrifuge bottles, acidified to pH 1 - 2 with concentrated HC1, and the resulting precipitates are collected by centrifugation (16,000 g). After discarding the supernatants, the pellets are washed once with acidic water and air dried at 50 ° for 24 to 48 hr. The weight of each precipitate is determined to _ 0.1 mg accuracy, and then the Klason lignin, carbohydrate, protein, and ash contents of each APPL are determined (see Chapter [5] in this volume). Using the data obtained from all of these analyses, a mg APPL versus 600 nm standard curve (corrected for protein contamination) can be constructed, and an APPL versus time production curve can be drawn and compared with curves showing the rates of lignin and carbohydrate depletion and the rate of actinomycete cell mass increase over time.
Additional Comments In all cases, APPLs derived from lignin should contain a high percentage (60-80%) of acid-insoluble component as measured by the Klason lignin assay, and they should also contain some carbohydrate (5-10%), mostly in the form of lignin-associated hemicelluloses still complexed with the solubilized lignin. 4 In addition, any APPL recovered should be offset by
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a similar or greater loss of lignin from the lignocellulosic substrate. 3,4 However, there is one problem that may be encountered with certain ligninolytic Streptomyces. If an organism produces a highly modified lignin-derived APPL, that APPL may not assay as high in lignin content as expected because it may contain considerable amounts of acid-soluble lignin. 4 To conclusively prove the lignin origin of the APPL, chemical characterization of the lignin by acidolysis, permanganate oxidation, and ester hydrolysis is required (see Chapter [5] in this volume). Confirmation of r - E t h e r Linkage Cleavage Activity Another procedure for confirming lignin depolymerization is acidolysis, a chemical degradative procedure that shows whether or not there is a lower number offl-aryl ether bonds in degraded lignins as compared to the native lignin from which they were derived. 3,4 Conclusions are drawn from quantitative data on the amounts of two key acidolysis products. The amount of one product, phenylpropane Ketol I, should be lower in depolymerized lignins as compared to native lignin, while the amount of a second product, vanillic acid, should be greater. This procedure requires a sample of purified native lignin and degraded lignin from the lignocellulose substrate being used. Minimally, 5 g corn lignocellulose (solid-state or submerged culture fermentations) is incubated as described above for a sufficient length of time to ensure that substantial lignin degradation has occurred (4-6 weeks). The partially degraded lignocellulose residues and APPLs are then recovered as described above. Purified milled corn lignins (MCL) are prepared from the degraded lignocellulose and from an undegraded control lignocellulose using the neutral solvent extraction procedure of Bj6rkman 7 (see Chapter [3] in this volume). Then the APPL and the purified lignins are chemically degraded using the simplified acidolysis procedure of Pometto and Crawfords and the yields of Ketol I and vanillic acid obtained from each lignin and APPL are then compared. The results for the control MCL are considered as native lignin baseline values, and the yield of Ketol I should be considerably higher than the yield of vanillic acid. The ratio of Ketol I to vaniUic acid should, however, decrease for degraded lignins and APPLs. For example, values reported previously for control MCLs and for APPLs resulting from lignin depolymerization by S. viridosporus are 5.4/0.7% and 2.9/2.2%, respectively (Ketol I/vanillic acid yields, as a percentage of the lignin or APPL subjected to acidolysis).3 7A. Bj6rkman,Sven. Papperstidn. 59, 477 (1956). s A. L. PomettoIII and D. L. Crawford,Appl. Environ. Microbiol.49, 879 (1985).
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Acidolysis In this procedure, APPLs and BjSrkman lignins from decayed and control lignocelluloses are subjected to a 6-hr hydrolysis in acidic dioxane. 8 Hydrolyzed samples are then solvent extracted to recover single-ring acidolysis products which are in turn identified and quantified by gas-liquid chromatography (GLC) using procedures described in Chapter [ 16] of this volume. The work-up procedure is tedious and requires attention to detail. A solution of 15 mg of Bj0rkman lignin or APPL/ml of 0.2 M HC1 in dioxane- water (9: 1) is placed in a glass ampoule, which is then cooled in an ice bath, flushed with nitrogen, and flame sealed. The sealed ampoule is placed into a heating block at 87 ° for 6 hr. Glycerol is added to the block wells to promote more even heat transfer. After cooling, the ampoule is opened, diluted with water to give a final dioxane/water ratio of 1 : 1, and then quantitatively transferred to a separatory funnel using acidic dioxane-water (1 : 1) washes to ensure complete transfer. The acidolysis mixture is extracted four times with chloroform-acetone (1 : 1) and then once with chloroform only. Combined extracts are dried under a vacuum at 4 5 - 5 0 °, then the dry extracts are again extracted, this time with dioxane-chloroform (1:I), until extracts are colorless. The combined extracts are transferred into a preweighed beaker and evaporated to dryness in a hood at room temperature. Then the weight of recovered solids is determined. These residues are dissolved in ethyl acetate-ethanol 95% (1: 1) to a final concentration of 10 mg/ml. A 0.3-ml (=3 mg) sample is pipetted into a dry (predesiccated), preweighed vial, and the solution is evaporated to dryness in a hood. Next the vial is desiccated for 72 hr under nitrogen to avoid oxidation of any acidolysis products. The weight of the residue is then determined to _+0.1 mg accuracy. The products and known standards are next converted to their trimethylsilyl derivatives and each is then quantified by GLC (see Chapter [ 16] in this volume).
Other Comments Twelve important single-ring aromatic acidolysis products are typically recovered from softwood lignins after acidolysis. 8,9 Similar products, some with different ring substitution patterns, are recovered from hardwood and grass lignins.~ The most important and most dominant product found in nondegraded Bj6rkman lignin is Ketol I (1-hydroxy-3-[4-hydroxy-3-methoxyphenyl]-2-propanone), which is produced only when the fl-aryl ether bond of the polymer is intact at the time of acidolysis. When extensive depolymerization has occurred, vanillic acid (4-hydroxy-3-methoxyben9 K. Lundquist and T. K. Kirk, Acta Chem. Scand. 25, 889 (1971).
[24]
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257
zoic acid) typically becomes a more dominant product, and the yield of Ketol I decreases. Liguins and APPLs from degraded lignoceUuloses will often have a Ketol I/vanillic acid ratio of less than 1 while the corresponding native lignin will have a ratio greater than I. 3,4 For example, with softwood lignins from spruce (Picea pungens) the ratio from undegraded BjOrkman lignin was reported to be 1.3 (4.2% Ketol I/3.2% vanillic acid), whereas the ratio from a lignin derived from S. viridosporus-degraded spruce lignocellulose was 0.5 (2.4% Ketol I/5.1% vanillic acid). I° Similarly, the ratio of Ketol I to vanillic acid for an S. viridosporus APPL derived from corn lignocellulose was 1.3 (2.9% Ketol I/2.2% vanillic acid) as compared to a ratio of 3.7 (5.2% Ketol I/1.4% vanillic acid) for the control MCL. 3 These results demonstrated that the APPL, though modified, was definitely derived from a true lignin polymer containing fl-aryl ether bonds. On the other hand, acidolysis can sometimes give unexpected results. For example, acidolysis of an S. badius APPL produced an acidolysis product mixture that was distinctly nonligninlike (yielding no Ketol I or vanillic acid), 4 an indication that this APPL was either not a lignin-derived product, or else it was so extensively modified by this actinomycete that it no longer resembled lignin. Assaying Previously Unstudied Organisms for LigninDepolymerizing/Solubilizing Activity The turbidometric assay for determination of solubilized polymeric lignin fragments can be utilized to screen a wide variety of microorganisms for lignin-depolymerizing ability. In general the assay is employed in the screening of cultures isolated previously by selection on lignocellulosecontaining media, or selected based upon the ability to mineralize [14C]lignin-labeled lignocellulosesI (see Chapter [3] on [14C]lignin degradation). In particular, the turbidometric assay will be useful for examining filamentous actinomycetes and fungi because these microbes do not produce turbidity since they grow filamentously. However, it is possible to study nonfilamentous bacteria if assay samples are centrifuged to remove the bacterial cells prior to the acidification step that precipitates the APPLs. Whenever a new organism is found to solubilize lignin, it will also be necessary to chemically characterize the APPL-like product to confirm its lignin origin, and to confirm a corresponding loss of lignin from the lignocellulose substrate. With the Streptomyces thus far studied, APPLs appear to be essentially lOD. L. Crawford, M. J. Barder, A. L. Pometto III, and R. L. Crawford, Arch. Microbiol. 131, 140 (1982).
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a terminal product of lignin metabolism, or at most an intermediate that is only slowly metabolized further. 4,6 However, some as yet undiscovered organisms may produce APPLs and then metabolize them rapidly. With these organisms, APPLs would be a transitory intermediate and would likely not accumulate in amounts equivalent to the lignin lost from the lignocellulose. For example, the white rot fungus Phanerochaete chrysosporium is capable of completely degrading lignin to CO2 and H20, but it produces only small amounts of an APPL-Iike intermediate when it is growing on lignocellulose.TM The chemistry of this product has not been extensively studied. Another factor to be considered is that the enzymatic mechanism for APPL production may be different for different microorganisms. For example, while the extracellular enzymes involved in the initial oxidation of lignin by P. chrysosporium have now been identified,H the enzymes responsible for APPL release by Streptomyces remain to be discovered and are probably quite different from the ligninases of P. chrysosporium) 2 This complexity of variable must always be considered when new cultures are being examined for ligninolytic activities. H R. L. Crawford and D. L. Crawford, Enzyme Microb. Technol. 6, 434 (1984). ~2 D. L. Crawford, A. L. Pometto III, and L. A. Deobald, "Recent Advances in Lignin Biodegradation Research," pp. 78-95. Uni Publ., Tokyo, 1983.
[25] Manganese
Peroxidase
from
P h a n e r o c h a e t e chrysosporiurn By MICHAEL H. GOLD and JEFFREY K. GLENN
2Mn(II) + H202 + 2H + --* 2Mn(III) + 2H20
Principle. Mn(II) peroxidase is an extracellular enzyme expressed during secondary metabolism as part of the lignin-degradative system of Phanerochaete chrysosporium. Mn(II) peroxidase oxidizes Mn(II) to Mn(III). Mn(III) is a nonspecific oxidant which in turn oxidizes a variety of organic compounds.~-a
M. Kuwahara, J. K. Glenn, M. A. Morgan, and M. H. Gold, FEBSLett. 169, 247 (1984). 2 j. K. Glenn and M. H. Gold, Arch. Biochem. Biophys. 242, 329 (1985). 3 A. Paszczyfiski, V.-B. Huynh, and R. Crawford, Arch. Biochem. Biophys. 244, 750 (1986).
METHODS IN ENZYMOLOGY, VOL. 161
Copyright© 1988by AcademicPress,Inc. All fishtsof reproductionin any formr~erved.
LIGNIN DEPOLYMERIZATION
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strated by ESR spectroscopy. Kersten et al. 9 detected the cation radicals of methoxybenzenes by ESR spectroscopy; Hammel et al. ~o detected radicals from dimeric model compounds oflignin through ESR spin-trapping techniques. Largely through the results of these two studies, a generalized mechanism for lignin degradation can be formulated. This generalized mechanism involves a central role for substrate aryl cation free radicals. Cation radicals can undergo a wide range of reactions; the type of reactions can be affected by the ring substituents. Substrates with a-hydroxy-containing propyl side chains (prominent in lignin) preferentially undergo carbon-carbon bond cleavage.t° Methoxybenzenes cation radicals tend to hydrate and demethylate for form formaldehyde and benzoquinones.9 Because the cation radicals are stable enough to diffuse away from the active site into the "bulk phase," their fate can also be dependent on the components of the bulk phase. Thus the pH, concentration of dioxygen, and concentration of other radicals can affect the addition of H20, addition of dioxygen, and dimerization with other radicals (reactions all observed with lignin models). Much of our understanding of the chemistry of cation radicals has been provided by Snook and Hamilton. 21 These workers studied the formation and degradation of aryl cation radicals in chemical systems. These studies have provided a model for ligninase catalysis; they have indicated that mechanistically, the chemistry of cation radicals accounts for most if not all of the prominent reactions observed in lignin biodegradation. Carboncarbon bond cleavage of propyl side chains, loss of methoxyls, oxidation of benzylic hydroxyls, and ring opening are mechanically consistent with a free radical mechanism. It is thus apparent that the future utilization of ligninase will require not only an understanding of ligninase catalysis, but also the chemistry of cation radicals. 21M. E. Snookand G. A. Hamilton,J. Am. Chem. Soc. 96, 860 (1974).
[24] L i g n i n - D e p o l y m e r i z i n g
Activity of Streptomyces
B y DON L. CRAWFORDand ANTHONY L. POMETTO III
Lignin is a complex phenylpropane polymer consisting of coumaryl, guaiacyl, and syringyl moieties linked together by numerous linkages, but primarily by the fl-aryl ether bond) Biodegradation of this recalcitrant R. L. Crawford, "Lignin Biodegradationand Transformation,"Wiley(Interscience),New
York, 1981. METHODS IN ENZYMOLOGY, VOL. 161
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form r--,;~erved.
250
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polymer by Streptomyces is by an oxidative process in which the ether bonds are cleaved and the phenylpropane side chains are modified or removed.2 Some low-molecular-weight aromatic intermediates are released from lignin as a result of these reactions, but once produced they may or may not be catabolized by specific Streptomyees. 2 A principal result of fl-aryl ether bond cleavage is the substantial depolymerization of lignin. While Streptomyces-mediated depolymerization results in the production of relatively small amounts of single-ring aromatic intermediates, it results in the release of large amounts of a microbially modified water-soluble lignin polymer that is precipitable from aqueous solutions when they are acidified. This polymer has been named acid-precipitable polymeric lignin (APPL). 3 APPLs are the principal initial product of lignin degradation by the ligninolytic Streptomyces that have been studied thus far? In this chapter, we describe a simple spectrophotometric assay for monitoring Streptomyces-mediated solubilization of lignin when cultures are degrading lignocellulosic substrates in submerged cultures or in solid-state fermentations. We also describe a simplified lignin acidolysis procedure which is important for chemically characterizing APPLs in order to establish their lignin origin, to confirm that fl-aryl ether bonds in the original lignin were cleaved, and to demonstrate that lignin depolymerization occurred. Both the spectrophotometric and chemical assays are valuable for use in screening for new lignocellulose-decomposing microorganisms having the ability to depolymerize and simultaneously solubilize lignin. Cultivation of Streptomyces on Lignocellulose The preparation of the lignocellulose substrate and of log phase cells to be used as inoculum in solid-state fermentation and submerged culture systems is described in Chapter [5] in this volume. In this discussion, solid-state fermentations are described for Streptomyces viridosporus T7A (ATCC No. 39115) and a submerged culture system is described for Streptomyces badius 252 (ATCC No. 39117). These Streptomyces are examples of the two known types of lignin-depolymerizing actinomycetes,4 either of which might be encountered when using the lignin-depolymerization assay.
Solid-State Fermentation By following the procedures outlined in Chapter [5], 5 ml of late log phase cells of S. riridosporus is inoculated into 0.5 g of sterile ground and 2 D. L. Crawford and R. L. Crawford, Enzyme Microb. Technol. 2, 11 (1980). 3 D. L. Crawford, A. L. Pometto III, and R. L. Crawford, Appl. Environ. Microbiol. 45, 898 (1983). 4 j. R. Borgraeyer and D. L. Crawford, Appl. Environ. Micrbiol. 49, 273 (1985).
[24]
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extracted corn (Zea mays) lignocellulose, and incubations are carried out in cotton-plugged 250-m1 flasks. Incubation is at 37 ° in a humid incubator. Sufficient replicates are incubated so that at least three flasks can be harvested at each time interval to be examined, and a similar number of controls containing sterile lignocellulose moistened with only sterile meditim are incubated as well. The number of samplings per experiment will vary with the metabolic rate of the organism being studied; however, in initial experiments flasks should probably be harvested in replicates of three at 4-day intervals. After inoculation, time zero inoculated and control flasks are retained for immediate assay, and the remaining flasks are placed into the incubator. To prevent moisture loss flasks can be placed in a plastic bag along with a beaker of water to ensure that a humid environment is maintained. At periodic time intervals, three inoculated and three control flasks are removed for assay. Each flask is assayed for its content of polymeric water-soluble lignin (APPL), using the turbidometric procedure described below. Results are reported as averages of three replicate values with standard deviations.
Submerged Culture System Following the procedures described in Chapter [5] in this volume, 50 ml of late log phase cells of S. badius is inoculated into 5 g of sterile ground and extracted corn lignocellulose in a 2-liter flask, and the culture is preincubated as a solid-state fermentation at 37 ° in a humid incubator for 3 to 4 days. At the end of this period, 1.0 liter of additional liquid medium [0.6% (w/v) yeast extract in mineral salts] is added. This medium consists of 6.0 g of yeast extract (Difco Laboratories, Detroit, MI), 5.3 g Na2HPO4, 1.98 g KH2PO4, 0.2 g MgSO4"7H20, 0.2 g NaC1, 0.05 g CaC12" 2H20, plus 1.0 ml of trace element solution (6.4 g CuSO4" 5H20, 1.1 g FeSO4"7H20, 7.9g MnC12"4H20, 1.5g ZnSO4"7H20/liter of water) 5 per liter of deionized H20, adjusted to pH 7.1 - 7.2. After addition of the liquid medium, the culture is shifted to shaking incubation at 100 rpm and 37 °, and cultures are incubated in replicates of three. Three uninoculated controls prepared using only sterile medium in place of cell suspension are also incubated, and these controls are carded through the same sequence of manipulations as were the inoculated cultures. A zero time 10-ml sample of culture medium is taken aseptically at the time that the liter of medium is added to each of the solid-state fermentation flasks. Additional 10-ml samples are taken at 2-day intervals. Each sample is then assayed for soluble lignin (APPL) by the turbidometric procedure described below, and results are reported as averages of three replicate values with standard deviations. 5 T. G. Pridham and G. Gottlieb, J. Bacteriol. 56, 107 (1948).
252
LIGNIN
[24]
Turbidometric Assay for Water-Soluble Polymeric Lignin F r a g m e n t s
Solid-State Fermentation To each solid-state fermentation flask to be assayed, 125 ml of distilled water is added. The flask is then placed in a boiling water bath or steamer for 1 hr, during which time it is shaken periodically. Next, the suspension is filtered through preweighed filter paper (Whatman No. 54) to recover the residual lignocellulose/biomass for substrate weight loss determinations and later chemical analysis of the lignocellulosic residue (see below). For spectrophotometric assay of APPL, a 2.0-ml portion of the filtrate is pipetted into a cuvette, 0.1 ml of concentrated HCI is added to acidify the sample, and the solution is thoroughly mixed. This acidified sample is allowed to stand at least 1 hr at 4°, over which time any APPL or other acid-precipitable material present will precipitate. The sample is then remixed, and its optical density (OD) is determined at 600 nm against a water blank. Several repeat readings are taken to minimize variation of data due to the settling of the precipitate during measurement. An average OD reading is then calculated. When a reading is taken, it is also advisable to calculate average values based upon three to four separate 2.0-ml sampies. By periodically harvesting flasks and performing turbidometric assays as described, a turbidity versus time plot can be drawn, and production of APPL by the culture over time can be monitored.
Submerged Culture System The assay for lignin solubilization by cells growing as submerged cultures is simpler than for those growing as solid-state fermentations since the cultures do not have to be subjected to the hot water extraction procedure. Ten-milliliter samples of culture supernatant are aseptically withdrawn from the incubating culture at 2-day intervals, and they are filtered to remove any insoluble material. The filtrate is then assayed for solubilized lignin turbidometrically at 600 nm as above.
Correctionfor Protein in the Precipitates The only disadvantage of relying solely on the turbidometric assay for following APPL production by Streptomyces is that extracellular proteins may also precipitate when samples are acidified, and thus inflate the APPL values. 6 This problem seems to be more evident in the submerged culture 6 A. L. Pometto III and D. L. Crawford, Appl. Environ. Microbiol. 51, 171 (1986).
[24]
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system than in the solid-state fermentation system. It is relatively easy to correct for protein contamination by assaying air-dried APPL precipitates for crude protein content by the Kjeldahl procedure (see below and Chapter [5] in this volume). Confirmation of Lignin-Solubilizing Activity If a specific microbial strain grows well on lignocellulose in solid-state or submerged culture and produces increasing amounts of APPL-like material over time as shown by the turbidometric assay, then it can be considered tentatively as a lignin-depolymerizing microorganism. The acid-precipitable material measured by the turbidometric assay must, however, be examined to confirm that it is lignin in origin and that a correlation exists such that the appearance of solubilized lignin corresponds to a similar pattern of loss of insoluble lignin from the lignocellulose substrate (see Chapter [5] in this volume). 4
Confirmation for Solid-State Fermentations It is desirable first to quantify the exact amount of APPL present in filtrates taken from the different-aged cultures discussed above. When each flask is harvested, the total volume of filtrate is determined, and then the filtrate is acidified to pH 1 - 2 with concentrated HC1. The precipitate that forms is collected by centrifugation (16,000 g) in preweighed centrifuge bottles. The supernatants are discarded. Each precipitate is washed once with acidic water, and then it is air dried at 50 ° for 24 to 48 hr. The weight of the total recovered APPL is next determined to ___0.1 mg accuracy, and with the total volume removed for turbidometric assay taken into consideration, the total milligram weight of APPL recovered from the culture is calculated. After the dry weight of APPL is determined, the Klason lignin, carbohydrate, protein, and ash contents of the APPL are determined (see Chapter [5]). From these data it is possible to correlate the actual weight of lignin-derived APPL with the OD60o readings, and thereby generate a standard APPL curve for the organism under study. The standard curve is presented as a graph of OD60o against mg/ml APPL values. One should also calculate the percentage weight loss of lignocellulosic substrate at each time interval by weighing the air-dried residues recovered at each filtration step. The crude protein, lignin, and carbohydrate contents of the insoluble residues can also be determined so that cell mass increases and lignin and carbohydrate depletion from the lignocellulosic substrate can be calculated and compared with the rate and extent of APPL production by specific ligninolytic Streptomyces (see Chapter [5] in this volume).
254
LIGNIN
[24]
Submerged Culture System Once the time course of apparent lignin solubilization has been determined, tentatively identified lignin-solubilizing cultures are again grown on lignocellulose as described above. However, this time the cultures should be grown in 250-ml flasks containing 0.5 g of lignocellulose, and three replicate flasks should be harvested at each time interval. Sterile uninoculated controls should be incubated in the same fashion. Preincubation as solid-state fermentations is used as before, which in this case requires an inoculum of 5 ml of late log cells (for S. badius), followed by 3 - 4 days of preincubation at 37 °. Then 100 ml of the 0.6% (w/v) yeast extract- mineral salts medium is added prior to aerobic shaking incubation at 100 rpm and 37 °. At the time of initiating the submerged culture portion of the incubation and at 2- to 3-day intervals thereafter, three flasks each from the inoculated cultures and the uninoculated sterile controls are harvested. The flasks are placed in a boiling water bath or steamer for 1 hr, and the residual lignocelluloses are recovered by filtration through preweighed filter paper. The residues are air dried and weighed as described above so that the rate of lignocellulose weight loss over time can be calculated. Each residue is also chemically analyzed for its crude protein, lignin, and carbohydrate contents (see Chapter [5] in this volume) so that increases in actinomycete cell mass and lignin and carbohydrate depletion rates can be calculated. The filtrates are transferred into preweighed centrifuge bottles, acidified to pH 1 - 2 with concentrated HC1, and the resulting precipitates are collected by centrifugation (16,000 g). After discarding the supernatants, the pellets are washed once with acidic water and air dried at 50 ° for 24 to 48 hr. The weight of each precipitate is determined to _ 0.1 mg accuracy, and then the Klason lignin, carbohydrate, protein, and ash contents of each APPL are determined (see Chapter [5] in this volume). Using the data obtained from all of these analyses, a mg APPL versus 600 nm standard curve (corrected for protein contamination) can be constructed, and an APPL versus time production curve can be drawn and compared with curves showing the rates of lignin and carbohydrate depletion and the rate of actinomycete cell mass increase over time.
Additional Comments In all cases, APPLs derived from lignin should contain a high percentage (60-80%) of acid-insoluble component as measured by the Klason lignin assay, and they should also contain some carbohydrate (5-10%), mostly in the form of lignin-associated hemicelluloses still complexed with the solubilized lignin. 4 In addition, any APPL recovered should be offset by
[24]
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a similar or greater loss of lignin from the lignocellulosic substrate. 3,4 However, there is one problem that may be encountered with certain ligninolytic Streptomyces. If an organism produces a highly modified lignin-derived APPL, that APPL may not assay as high in lignin content as expected because it may contain considerable amounts of acid-soluble lignin. 4 To conclusively prove the lignin origin of the APPL, chemical characterization of the lignin by acidolysis, permanganate oxidation, and ester hydrolysis is required (see Chapter [5] in this volume). Confirmation of r - E t h e r Linkage Cleavage Activity Another procedure for confirming lignin depolymerization is acidolysis, a chemical degradative procedure that shows whether or not there is a lower number offl-aryl ether bonds in degraded lignins as compared to the native lignin from which they were derived. 3,4 Conclusions are drawn from quantitative data on the amounts of two key acidolysis products. The amount of one product, phenylpropane Ketol I, should be lower in depolymerized lignins as compared to native lignin, while the amount of a second product, vanillic acid, should be greater. This procedure requires a sample of purified native lignin and degraded lignin from the lignocellulose substrate being used. Minimally, 5 g corn lignocellulose (solid-state or submerged culture fermentations) is incubated as described above for a sufficient length of time to ensure that substantial lignin degradation has occurred (4-6 weeks). The partially degraded lignocellulose residues and APPLs are then recovered as described above. Purified milled corn lignins (MCL) are prepared from the degraded lignocellulose and from an undegraded control lignocellulose using the neutral solvent extraction procedure of Bj6rkman 7 (see Chapter [3] in this volume). Then the APPL and the purified lignins are chemically degraded using the simplified acidolysis procedure of Pometto and Crawfords and the yields of Ketol I and vanillic acid obtained from each lignin and APPL are then compared. The results for the control MCL are considered as native lignin baseline values, and the yield of Ketol I should be considerably higher than the yield of vanillic acid. The ratio of Ketol I to vaniUic acid should, however, decrease for degraded lignins and APPLs. For example, values reported previously for control MCLs and for APPLs resulting from lignin depolymerization by S. viridosporus are 5.4/0.7% and 2.9/2.2%, respectively (Ketol I/vanillic acid yields, as a percentage of the lignin or APPL subjected to acidolysis).3 7A. Bj6rkman,Sven. Papperstidn. 59, 477 (1956). s A. L. PomettoIII and D. L. Crawford,Appl. Environ. Microbiol.49, 879 (1985).
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[24]
Acidolysis In this procedure, APPLs and BjSrkman lignins from decayed and control lignocelluloses are subjected to a 6-hr hydrolysis in acidic dioxane. 8 Hydrolyzed samples are then solvent extracted to recover single-ring acidolysis products which are in turn identified and quantified by gas-liquid chromatography (GLC) using procedures described in Chapter [ 16] of this volume. The work-up procedure is tedious and requires attention to detail. A solution of 15 mg of Bj0rkman lignin or APPL/ml of 0.2 M HC1 in dioxane- water (9: 1) is placed in a glass ampoule, which is then cooled in an ice bath, flushed with nitrogen, and flame sealed. The sealed ampoule is placed into a heating block at 87 ° for 6 hr. Glycerol is added to the block wells to promote more even heat transfer. After cooling, the ampoule is opened, diluted with water to give a final dioxane/water ratio of 1 : 1, and then quantitatively transferred to a separatory funnel using acidic dioxane-water (1 : 1) washes to ensure complete transfer. The acidolysis mixture is extracted four times with chloroform-acetone (1 : 1) and then once with chloroform only. Combined extracts are dried under a vacuum at 4 5 - 5 0 °, then the dry extracts are again extracted, this time with dioxane-chloroform (1:I), until extracts are colorless. The combined extracts are transferred into a preweighed beaker and evaporated to dryness in a hood at room temperature. Then the weight of recovered solids is determined. These residues are dissolved in ethyl acetate-ethanol 95% (1: 1) to a final concentration of 10 mg/ml. A 0.3-ml (=3 mg) sample is pipetted into a dry (predesiccated), preweighed vial, and the solution is evaporated to dryness in a hood. Next the vial is desiccated for 72 hr under nitrogen to avoid oxidation of any acidolysis products. The weight of the residue is then determined to _+0.1 mg accuracy. The products and known standards are next converted to their trimethylsilyl derivatives and each is then quantified by GLC (see Chapter [ 16] in this volume).
Other Comments Twelve important single-ring aromatic acidolysis products are typically recovered from softwood lignins after acidolysis. 8,9 Similar products, some with different ring substitution patterns, are recovered from hardwood and grass lignins.~ The most important and most dominant product found in nondegraded Bj6rkman lignin is Ketol I (1-hydroxy-3-[4-hydroxy-3-methoxyphenyl]-2-propanone), which is produced only when the fl-aryl ether bond of the polymer is intact at the time of acidolysis. When extensive depolymerization has occurred, vanillic acid (4-hydroxy-3-methoxyben9 K. Lundquist and T. K. Kirk, Acta Chem. Scand. 25, 889 (1971).
[24]
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zoic acid) typically becomes a more dominant product, and the yield of Ketol I decreases. Liguins and APPLs from degraded lignoceUuloses will often have a Ketol I/vanillic acid ratio of less than 1 while the corresponding native lignin will have a ratio greater than I. 3,4 For example, with softwood lignins from spruce (Picea pungens) the ratio from undegraded BjOrkman lignin was reported to be 1.3 (4.2% Ketol I/3.2% vanillic acid), whereas the ratio from a lignin derived from S. viridosporus-degraded spruce lignocellulose was 0.5 (2.4% Ketol I/5.1% vanillic acid). I° Similarly, the ratio of Ketol I to vanillic acid for an S. viridosporus APPL derived from corn lignocellulose was 1.3 (2.9% Ketol I/2.2% vanillic acid) as compared to a ratio of 3.7 (5.2% Ketol I/1.4% vanillic acid) for the control MCL. 3 These results demonstrated that the APPL, though modified, was definitely derived from a true lignin polymer containing fl-aryl ether bonds. On the other hand, acidolysis can sometimes give unexpected results. For example, acidolysis of an S. badius APPL produced an acidolysis product mixture that was distinctly nonligninlike (yielding no Ketol I or vanillic acid), 4 an indication that this APPL was either not a lignin-derived product, or else it was so extensively modified by this actinomycete that it no longer resembled lignin. Assaying Previously Unstudied Organisms for LigninDepolymerizing/Solubilizing Activity The turbidometric assay for determination of solubilized polymeric lignin fragments can be utilized to screen a wide variety of microorganisms for lignin-depolymerizing ability. In general the assay is employed in the screening of cultures isolated previously by selection on lignocellulosecontaining media, or selected based upon the ability to mineralize [14C]lignin-labeled lignocellulosesI (see Chapter [3] on [14C]lignin degradation). In particular, the turbidometric assay will be useful for examining filamentous actinomycetes and fungi because these microbes do not produce turbidity since they grow filamentously. However, it is possible to study nonfilamentous bacteria if assay samples are centrifuged to remove the bacterial cells prior to the acidification step that precipitates the APPLs. Whenever a new organism is found to solubilize lignin, it will also be necessary to chemically characterize the APPL-like product to confirm its lignin origin, and to confirm a corresponding loss of lignin from the lignocellulose substrate. With the Streptomyces thus far studied, APPLs appear to be essentially lOD. L. Crawford, M. J. Barder, A. L. Pometto III, and R. L. Crawford, Arch. Microbiol. 131, 140 (1982).
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a terminal product of lignin metabolism, or at most an intermediate that is only slowly metabolized further. 4,6 However, some as yet undiscovered organisms may produce APPLs and then metabolize them rapidly. With these organisms, APPLs would be a transitory intermediate and would likely not accumulate in amounts equivalent to the lignin lost from the lignocellulose. For example, the white rot fungus Phanerochaete chrysosporium is capable of completely degrading lignin to CO2 and H20, but it produces only small amounts of an APPL-Iike intermediate when it is growing on lignocellulose.TM The chemistry of this product has not been extensively studied. Another factor to be considered is that the enzymatic mechanism for APPL production may be different for different microorganisms. For example, while the extracellular enzymes involved in the initial oxidation of lignin by P. chrysosporium have now been identified,H the enzymes responsible for APPL release by Streptomyces remain to be discovered and are probably quite different from the ligninases of P. chrysosporium) 2 This complexity of variable must always be considered when new cultures are being examined for ligninolytic activities. H R. L. Crawford and D. L. Crawford, Enzyme Microb. Technol. 6, 434 (1984). ~2 D. L. Crawford, A. L. Pometto III, and L. A. Deobald, "Recent Advances in Lignin Biodegradation Research," pp. 78-95. Uni Publ., Tokyo, 1983.
[25] Manganese
Peroxidase
from
P h a n e r o c h a e t e chrysosporiurn By MICHAEL H. GOLD and JEFFREY K. GLENN
2Mn(II) + H202 + 2H + --* 2Mn(III) + 2H20
Principle. Mn(II) peroxidase is an extracellular enzyme expressed during secondary metabolism as part of the lignin-degradative system of Phanerochaete chrysosporium. Mn(II) peroxidase oxidizes Mn(II) to Mn(III). Mn(III) is a nonspecific oxidant which in turn oxidizes a variety of organic compounds.~-a
M. Kuwahara, J. K. Glenn, M. A. Morgan, and M. H. Gold, FEBSLett. 169, 247 (1984). 2 j. K. Glenn and M. H. Gold, Arch. Biochem. Biophys. 242, 329 (1985). 3 A. Paszczyfiski, V.-B. Huynh, and R. Crawford, Arch. Biochem. Biophys. 244, 750 (1986).
METHODS IN ENZYMOLOGY, VOL. 161
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