Vol. 47, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1984, p. 998-10040099-2240/84/050998-07$02.00/0Copyright ©D 1984, American Society for Microbiology

Anaerobic Biodegradation of the Lignin and PolysaccharideComponents of Lignocellulose and Synthetic Lignin by Sediment

MicrofloratRONALD BENNER,* A. E. MACCUBBIN,t AND ROBERT E. HODSON

Department of Microbiology and Institute of Ecology, University of Georgia, Athens, Georgia 30602

Received 3 January 1984/Accepted 14 February 1984

Specifically radiolabeled [14C-lignin]lignocelluloses and [14C-polysaccharide]lignocelluloses were pre-

pared from a variety of marine and freshwater wetland plants including a grass, a sedge, a rush, and a

hardwood. These [14C]lignocellulose preparations and synthetic [14C]lignin were incubated anaerobicallywith anoxic sediments collected from a salt marsh, a freshwater marsh, and a mangrove swamp. Duringlong-term incubations lasting up to 300 days, the lignin and polysaccharide components of the lignocellu-loses were slowly degraded anaerobically to 14CO2 and 14CH4. Lignocelluloses derived from herbaceousplants were degraded more rapidly than lignocellulose derived from the hardwood. After 294 days, 16.9% ofthe lignin component and 30.0% of the polysaccharide component of lignocellulose derived from the grass

used (Spartina alterniflora) were degraded to gaseous end products. In contrast, after 246 days, only 1.5%of the lignin component and 4.1% of the polysaccharide component of lignocellulose derived from thehardwood used (Rhizophora mangle) were degraded to gaseous end products. Synthetic [14C]lignin was

degraded anaerobically faster than the lignin component of the hardwood lignocellulose; after 276 days,3.7% of the synthetic lignin was degraded to gaseous end products. Contrary to previous reports, theseresults demonstrate that lignin and lignified plant tissues are biodegradable in the absence of oxygen.

Although lignocelluloses are recalcitrant to anaerobic biodegradation, rates of degradation measured inaquatic sediments are significant and have important implications for the biospheric cycling of carbon fromthese abundant biopolymers.

Lignocellulosic detritus derived from vascular plants is aquantitatively significant source of particulate organic mat-ter in wetland ecosystems such as marshes and swamps (3,17, 23). Generally, between 50 and 80% of aquatic plantbiomass is lignocellulose in which the lignin-to-polysaccha-ride ratios vary between 1:2 and 1:7 (17, 18, 22). Higher plantprimary production in wetland ecosystems is among thehighest known for natural environments. In Georgia saltmarshes, for instance, total annual aboveground and below-ground production has been estimated to be 2,300 to 2,800 gofC per m2 (25). Sediments in these organically rich environ-ments are usually waterlogged and anoxic within a centime-ter or less of the sediment-water interface. Therefore, muchof the organic detritus produced in the aerobic water columnis probably degraded by anaerobic microorganisms in thesediments. In addition to the large quantities of organicdetritus that settle onto the sediments from the water col-umn, more organic material is produced in situ in the anoxiczone of the sediments as belowground plant parts such asroots and rhizomes. Belowground production by wetlandplants such as Spartina alterniflora and Juncus roemerianusequals aboveground production and frequently is consideredgreater (19, 25).

In light of the high percentage of lignocellulosic detritusthat is produced in or sinks into highly reducing, anoxicsediments in wetlands, no model of carbon and energy flow

* Corresponding author.t Contribution no. 505 of the Marine Institute, University of

Georgia, Sapelo Island, GA 31327. Okefenokee Ecosystem Publica-tion no. 47.

t Present address: Department of Experimental Biology, RoswellPark Memorial Institute, Buffalo, NY 14263.

through such detritus-based ecosystems is complete withoutconsideration of rates and efficiencies of microbial transfor-mations of lignocellulose under anaerobic conditions. How-ever, it has been difficult to study these rates because nomethods existed which were sufficiently sensitive and selec-tive to individually track the biodegradation of the lignin orpolysaccharide components of natural lignocelluloses (7, 8).The recent introduction of radioisotopic methods has

greatly increased the accuracy and sensitivity of lignin andlignocellulose biodegradation assays. Several types of natu-ral and synthetic (dehydrogenation polymerizates [DHPs])14C-labeled lignin and lignocellulose preparations have beenused, and the advantages and limitations of each have beenreviewed (7). Investigators using these radiolabeled prepara-tions in studies of the aerobic biodegradation of lignin andlignocellulose have made much progress in determining ratesof degradation, the environmental factors affecting degrada-tion, and the microorganisms involved (7, 17, 21). However,there have been relatively few studies of the anaerobicbiodegradation of radiolabeled lignin and lignocellulosepreparations. Anaerobic degradation of lignin has been ob-served only in the alkaline digestive tract of some termites(5, 12), whereas no degradation of synthetic [14C]lignin or[14C-lignin]lignocellulose derived from poplar wood has beendetected in a variety of anoxic soils and sediments (13, 24,31). The results of these studies have led to the general beliefthat lignin is inert to biodegradation in anaerobic environ-ments.

Recalcitrance of lignin and lignocellulose in anaerobicenvironments has important implications for the biosphericcycling of carbon. The recalcitrant nature of lignocellulosicplant matter in anoxic sediments is frequently cited as amajor factor in the accumulation of peat and, eventually,coal (4). Studies of the physiology of white-rot fungi, impor-

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ANAEROBIC BIODEGRADATION OF LIGNIN 999

tant decomposers of lignin and lignocellulose in terrestrialenvironments, indicate that lignin degradation by theseorganisms is highly oxidative and may involve chemicaloxidants such as singlet oxygen and hydroxyl radicals (10,

11, 14). However, several investigators have demonstratedthe anaerobic degradation to methane and carbon dioxide ofmonomeric aromatic compounds, including those derivedfrom lignin, indicating that molecular oxygen is not required

for cleavage of the aromatic ring structures in lignin (9, 15,16, 28, 29). Since oxygen is not required for biodegradationof lignin-derived monomers, it appeared that molecularoxygen was required for cleavage of the intermonomericlinkages present in lignins. However, recent reports of the

anaerobic biodegradation of low-molecular-weight chemical-ly modified lignins and a lignin-related dimer containing the,B-aryl ether bond cast doubt on the requirement of molecularoxygen for polymer cleavage (6, 31).To investigate the anaerobic decomposition of the lignin

and polysaccharide components of lignocellulosic detritus,we specifically radiolabeled [14C-lignin]lignocelluloses and

[14C-polysaccharide]lignocelluloses from indigenous plantsof three distinct aquatic ecosystems: a salt marsh, an acidic(pH 3.8) freshwater wetland, and a mangrove swamp (1).Radiolabeled lignocelluloses derived from salt-marsh cord-grass (S. alterniflora) and needlerush (J. roemerianus),freshwater sedge (Carex walteriana), and red mangrove

(Rhizophra mangle) were incubated anaerobically with an-

oxic sediments from the environments where these plantswere collected. Synthetic [14C]lignin (DHPs) was also incu-bated anaerobically with anoxic sediments from the saltmarsh and the Okefenokee Swamp for comparative pur-

poses. All of the [14C]lignocellulose and synthetic [14C]ligninpreparations were slowly degraded anaerobically. Contraryto previous reports, lignin was degraded anaerobically tovarious extents depending on its source.

MATERIALS AND METHODSSampling sites and procedures. Anoxic sediments were

collected from a salt marsh on Sapelo Island, Ga., theOkefenokee Swamp in southern Georgia, and a mangrove

swamp at Fresh Creek estuary on Andros Island, Bahamas,during a cruise aboard O.R.V. Cape Florida in October 1982.Sediments from the salt marsh and the mangrove swamp

were sampled with sections of plastic (polyvinyl chloride)pipe (1 m by 5.5 cm). Cores were immediately sealed withbutyl rubber stoppers and transported to the laboratory atSapelo Island or aboard O.R.V. Cape Florida for processingwithin 4 h of collection. Sediment redox potential was

measured with an Orion Research model 901 specific ionmeter equipped with a platinum redox electrode. At a depthof 10 cm in a core from a J. roemerianus marsh on SapeloIsland, the redox potential was -289 mV, and the tempera-ture at this depth in the marsh was 30.5°C during August1982. At a depth of 10 cm in a core from a short-form S.alterniflora marsh on Sapelo Island, the redox potential was

-400 mV, and the temperature at this depth in the marshwas 310C during August 1982. At a depth of 1 cm in cores

from both marsh areas, the redox potential was <-150 mV.Cores were also collected from a red mangrove stand on

Andros Island, Bahamas, where the water depth was 1 m,

the salinity was 34%,o, the sediment temperature was 29°C,and the redox potential at a depth of 10 cm was -380 mV. Itwas not possible to obtain an intact core from OkefenokeeSwamp sediments because of the spongelike nature of thepeat, which is several meters deep. Samples of anoxic peatwere collected during November 1982 in a glass bottle from

Mizell Praire, an area dominated by the sedge C. walteriana.The water depth was 50 cm, the sediment temperature was28.5°C, the pH was 3.7, and the redox potential was -131mV.

Radiolabeled lignocelluloses and synthetic lignin. The pro-cedures used for radiolabeling the lignin and polysaccharidecomponents of lignocellulose derived from S. alterniflora, J.roemerianus, C. walteriana, and R. mangle have beenthoroughly described previously (1). Cuttings from livingplants were incubated for 3 days in sterile water containing aradioactive precursor of lignin or polysaccharides. The lignincomponent of the grass, rush, sedge, and mangrove leaveswas specifically radiolabeled with side chain [3-14C]cinnamicacid. Benner et al. (1) found, when using cinnamic acid tolabel the lignin component of herbaceous plants, that onlyminimal amounts (1 to 3%) of the label became associatedwith protein. During aerobic biodegradation studies, pepsin-treated (to remove contaminating protein), cinnamic acid-labeled lignocelluloses were found to be mineralized at thesame rate as cinnamic acid-labeled lignocelluloses which hadnot been pepsin treated, indicating that there was no interfer-ence from labeled protein when determining lignin degrada-tion rates (1). Studies of the pathways of anaerobic biodegra-dation of aromatic monomers, such as those derived fromlignin, have demonstrated that reduction of the aromatic ringstructure is required before removal of the side chain carbonadjacent to the aromatic ring (9, 16, 28). Thus, anaerobicbiodegradation of the cinnamic acid-labeled [14C-lignin]lig-nocelluloses used in this study would be indicative ofcleavage of the aromatic nucleus. Nitrobenzene oxidationand chromatographic analysis of the lignin-derived mono-mers from S. alterniflora [14C-lignin]lignocellulose revealedthat the specific activity of the three monomeric subunits issimilar (1). Thus, the radiolabel was distributed throughoutthe lignin macromolecule, suggesting that rates of degrada-tion of the radiolabel would be indicative of the overall lignindegradation rate. Mangrove wood was specifically radiola-beled in the lignin component with L-[U-14C]phenylalanine.When phenylalanine is used to specifically radiolabel thelignin component of mangrove wood, very little (<1%) of thelabel becomes associated with contaminating protein (1).Cuttings from all plants were preferentially labeled in thepolysaccharide component with D-[U-14C]glucose, whichlabels both cellulose and hemicellulose (1). After incorpo-ration of the radiolabel, the plant material was ground (<425,um), and an extract-free lignocellulose fraction was separat-ed from unincorporated label and other plant components byserial extractions in a soxhlet apparatus with boilingethanol (95%), ethanol-benzene (1:2 [vol/vol]), and water (1).Specific activities and detailed chemical analyses of thedistribution of label in the [14C-lignin]lignocelluloses and[14C-polysaccharide]lignocelluloses used in this study havebeen presented previously (1).

Synthetic [14C]lignins (DHPs) were kindly supplied byT. K. Kirk (U.S. Department of Agriculture Forest ProductsLaboratory, Madison, Wis.) and were uniformly radiola-beled in the carbon atoms of aromatic ring structures (20).The specific activity of the synthetic [14C]lignin was 47,600dpm/mg, and ca. 2 mg were used per incubation.

Anaerobic incubation procedures. Sections of sedimentcores at a depth of 5 to 10 cm were removed and placed in a

Waring blender specially equipped with gassing ports so thata continuous flow of N2 was maintained in the blender.Oxygen was removed from the N2 gas by passage over a

column of reduced copper filings heated to 4500C. An equalvolume of filter-sterilized (pore size, 0.2 [Lm; Nuclepore

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filter) deoxygenated water, collected from the site fromwhich the cores were taken, was added to the blender. Thesediments were homogenized (1 min), and 20-ml aliquotswere removed from the blender through a sampling port anddispensed into 35-ml serum bottles containing 20 mg of[14C]lignocellulose. The serum bottles were stoppered withbutyl rubber stoppers (Bellco Glass, Inc., Vineland, N.J.),aluminum crimp seals were attached, and the bottles wereflushed with 02-free N2 for 5 min. Controls were killed with10% Formalin (vol/vol, final concentration). The bottleswere incubated in anaerobic jars (GasPak; BBL Microbiolo-gy Systems, Cockeysville, Md.) at 30°C in the dark. Theprocedures for handling anoxic sediments incubated withsynthetic lignin were similar, except that all manipulationswere carried out in an anaerobic chamber (Coy LaboratoryProducts Inc., Ann Arbor, Mich.) and the bottles wereincubated in the chamber at room temperature (21 to 25°C).Production of "CO2 and 14CH4 was determined at 10- to

120-day intervals by flushing (with 02-free N2, 120 ml/minfor 4 min) the entire headspace of each serum bottle throughtwo carbon dioxide traps, each containing 10 ml of 1 NNaOH, and then into a Biological Oxidizer (R. J. HarveyInstrument Corp., Hillsdale, N.J.) to combust 14CH4 to14CO2, which was then trapped in liquid scintillation medium(1). The two NaOH traps were acidified (pH < 2) with 18 NH2SO4 and flushed into a series of two vials containing C02-trapping liquid scintillation medium. The recovery of 14CO2was >95%, as determined by an H14CO3 standard. By usingthis system to quantify radioactivity in evolved gases, verylow levels of degradation could be detected. In all incuba-tions with [14C]lignin-labeled substrates, conversion of 0.2%or less of the total added radioactivity was detectable asgaseous end products.To verify that the radioactivity being measured was asso-

ciated with 14CO2 and 14CH4, 500 ,ul of the headspace fromseveral incubations was removed with a gas-tight syringeand analyzed for 14CO2 and 14CH4 by separating thesecompounds via gas chromatography and collecting the puri-fied gases as they emerged from the column. Total carbondioxide and methane were measured with a thermal conduc-tivity detector and a 5-m Porapak Q column operated at 50°Cwith a He flow rate of 24 ml/min. Methane had a retentiontime of 2.7 min and was collected by water displacement.Radioactivity in the methane was determined by flushing themethane through a combustion oven (as described above)and trapping the evolved 14CO2 in a liquid scintillationmedium. Carbon dioxide had a retention time of 4.7 min andwas collected directly in a liquid scintillation medium. Theratio of 14CH4/14CO2 determined by this method was almostidentical to the ratio of 14CH4/14CO2 determined by flushingthe entire headspace through NaOH and then through acombustion oven as described above. Ratios of 14CH4/14CO2recovered from incubations with marine and freshwatersediments were similar (±+10%) to the ratios of total meth-ane/total carbon dioxide in the incubations as measured bygas chromatography.Upon termination of the incubations, the contents of each

serum bottle were analyzed for the presence of solubilizedradiolabel. Sediment slurries were washed from the serumbottles with sterile water (25 ml of 2% saline [pH 9], ordistilled water for Okefenokee samples) into 50-ml centrifugetubes. The tubes were vortexed and centrifuged (2,100 x g)for 10 min. The supernatant was decanted, and severalmilliliters from each incubation were filtered (pore size, 0.2or 0.45 ,Lm; Gelman filters). The total soluble radiolabel wasdetermined by adding 1 ml of each filtrate to a liquid

scintillation medium and quantifying the radioactivity byliquid scintillation spectrometry (1). Soluble radiolabeledorganic material (acid stable) was determined by acidifying(pH < 2) 1 ml of each filtrate with 1 N H2SO4 and assayingfor radioactivity as described above.

RESULTSDuring long-term anaerobic incubations with salt-marsh

sediments, the production of 14CO2 and 14CH4 from biodeg-radation of both the lignin and polysaccharide componentsof S. alterniflora and J. roemerianus [14C]lignocelluloseswas monitored (Fig. 1). After 294 days, 16.9% of the lignincomponent of S. alterniflora lignocellulose was degraded to14CO2 and 14CH4, whereas only 3.5% of J. roemerianuslignin was recovered as 14CO2 and 14CH4. A much greaterpercentage of the polysaccharide components was degraded:30% of the polysaccharide component of S. alternifloralignocellulose and 24% of the polysaccharide component ofJ. roemerianus lignocellulose were recovered as 14CO2 and14CH4 (Fig. 1). During the initial 33 days of incubation, nolabeled methane was detected. Analyses for labeled methanewere not conducted again until after 174 days of incubation.Within the incubation period, from day 117 to 174, labeledmethane and carbon dioxide were recovered in a ratio of 3:1,suggesting that as the incubations became depleted in sul-fate, methanogenesis became increasingly important. Whenthe additional label recovered as methane was considered,the calculated rates of decomposition of both the lignin andthe polysaccharide components of S. alterniflora and J.roemerianus lignocelluloses increased significantly, indicat-ing that decomposition rates were underestimated whenbased on the data from 33 to 117, during which only 14CO2evolution was monitored. A total of 27.3 and 18.9% of S.alterniflora and J. roemerianus lignocelluloses, respectively,was recovered as carbon dioxide and methane. These valuesare calculated from chemical analyses of the total percent-ages of lignin and polysaccharide in these lignocellulosepreparations. S. alterniflora lignocellulose is made up of80%polysaccharide and 20% lignin; J. roemerianus lignocellu-lose is made up of 75% polysaccharide and 25% lignin (17,22).

Specifically radiolabeled lignocellulose from the sedge C.walteriana, a common macrophyte in the OkefenokeeSwamp, was degraded relatively rapidly in the highly acidic,anaerobic peat (Fig. 2). Radiolabeled methane and carbondioxide were recovered in the ratio 2.3:1, respectively, from

CMN

0 30 - S. a/ternifloraC0) --J. roemerianuvs

o 4-o.

c- 12-

C) 6

50 100 150 200 250 300Incubation Time (Days)

FIG. 1. Anaerobic biodegradation of ["4C-lignin]lignocellulose(O) and [14C-polysaccharide]lignocellulose (0) in anoxic salt-marshsediments at 300C.

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ANAEROBIC BIODEGRADATION OF LIGNIN 1001

NC*_ 30 - 4%label recovered as 14Co2 + 14CH

~ 24 --% label recovered as 14CH4U 180

l2 -.

O 6

O 50 100 150 200 250 300Incubation Time (Days)

FIG. 2. Anaerobic biodegradation of C. walteriana [14C-lig-nin]lignocellulose (0) and [14C-polysaccharide]lignocellulose (0) inanoxic Okefenokee Swamp peat at 30°C.

both the lignin and the polysaccharide components of C.walteriana lignocellulose. A total of 9.8% of the lignincomponent and 20.1% of the polysaccharide component ofC. walteriana lignocellulose was degraded to 14CH4 and14C02 after 280 days. Based on a composition of 86%polysaccharide and 14% lignin (18), a total of 18.7% of thelignocellulose from C. walteriana was degraded to methaneand carbon dioxide.

Specifically radiolabeled lignocelluloses from the red man-grove, R. mangle, were very resistant to anaerobic biodegra-dation (Fig. 3). After 246 days, 1.4% of the lignin componentand 4.7% of the polysaccharide component of lignocellulosederived from mangrove leaves was recovered as 14CO2. Atotal of 1.5% of the lignin comnponent and 4.1% of thepolysaccharide component of lignocellulose derived frommangrove wood was recovered as 14C02. Gas chromato-graphic analyses of the headspace of incubations detectedonly minor amounts of methane (CO2:CH4 > 75:1); there-fore, incubations of sediments from the mangrove swampwere not assayed for 14CH4 production. Based on a composi-tion of 66% polysaccharide and 34% lignin (unpublisheddata), a total of 3.6% of the lignocellulose from mangroveleaves was recovered as carbon dioxide. Based on a compo-sition of 80% polysaccharide and 20% lignin (unpublisheddata), a total of 3.6% of the lignocellulose from mangrovewood was recovered as carbon dioxide.The anaerobic biodegradation of radiolabeled synthetic

c,J00 5 leoves

d! ~~wood04

0 0

50 100 150 200 250Incubation Time (Days)

FIG. 3. Anaerobic biodegradation of R. mangle [14C-lignin]ligno-cellulose (0) and ['4C-polysaccharide]lignocellulose (0) in anoxicmangrove sediments at 300C.

40 80 120 160 200 240Incubation Time (Days)

FIG. 4. Anaerobic biodegradation of synthetic ['4C]lignin in an-oxic salt-marsh sediments and Okefenokee Swamp peat at roomtemperature (21 to 25°C).

lignin (DHPs) in salt-marsh and Okefenokee Swamp sedi-ments is presented in Fig. 4. After 276 days of incubation,3.7% of synthetic [14C]lignin (DHPs) incubated in anoxicsalt-marsh sediments was degraded to 14CO2 and 14CH4.During the same incubation period, 1.7% of the DHPsincubated in anoxic Okefenokee Swamp peat was degradedto 14C02 and 14CH4. Rates of degradation in both salt-marshand Okefenokee Swamp sediments were continuously in-creasing during the first 162 days of incubation. The de-crease in rates of degradation after 160 days of incubationmay be an artifact of the long incubation period or may resultfrom increasing recalcitrance of the DHPs to biodegradation.An assessment of the percentage of radiolabel associated

with 14CO2 and 14CH4 is only a partial estimate of degrada-tion. Solubilized organic and inorganic compounds andcarbon incorporated into microbial biomass may constitutean important fraction of the total substrate degraded. Thepercentages of radiolabel recovered in soluble degradationproducts' after termination of the various incubations arepresented in Table 1. A total of 2.1 to 10.3% of theradioactivity from [14C-lignin]lignocelluloses was recoveredin soluble form. Likewise, a total of 0.2 to 12.7% of theradioactivity from [14C-polysaccharide]lignocelluloses wasrecovered in soluble form. When the additional radiolabelrecovered in soluble form from incubations of ["'Cilignocel-luloses from S. alterniflora and J. roemerianus was takeninto account, the calculated total percentage of degradationincreased by 29 to 65%. Lower percentages of the radiolabelwere recovered as soluble material from incubations ofOkefenokee Swamp sediments. This may be due to the lowpH, which would volatilize a greater percentage of solubleinorganics such as ["'C]bicarbonate or soluble organics suchas [14C]acetic acid. The inclusion of soluble radiolabel frommangrove swamp incubations increased the calculated per-centages of total [14C]ilignocellulose degradation by 160 to281%. The anaerobic incubations containing DHPs are stillin progress and have not been analyzed for soluble radiola-bel.The aerobic rates of degradation of the radiolabeled ligno-

celluloses and DHPs used in this study have been previouslydetermined (1, 17, 18; unpublished data). Synthetic ligninand lignocelluloses from marine intertidal plants were de-graded anaerobically at rates from 1 to 10% of the aerobicrates (Table 2). C. walteriana lignocellulose, incubated in

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TABLE 1. Radiolabel (%) from various [14C]lignocellulosesrecovered as CH4 plus C02, dissolved organic (acid-stable)material, total dissolved material, and total end products

% Radioactivity recovered as:

Lignocellulose CH4 Dissolved Total Totalplus organic dissolved decompositionCO2 material material

S. alternifloraLignin 16.9 4.2 10.3 27.2KCa 0 0.7 0.7 0.7Polysaccharide 29.9 4.2 12.7 42.7KC 0 2.6 2.6 2.6

J. roemerianusLignin 3.5 1.2 2.3 5.7KC 0 0.5 0.6 0.6Polysaccharide 24.0 3.2 7.0 31.0KC 0 2.1 2.3 2.3

C. walterianaLignin 9.8 1.9 2.1 11.9KC 0 0.4 1.3 1.3Polysaccharide 20.1 0.2 0.2 20.3KC 0 2.7 2.7 2.7

R. mangle (leaves)Lignin 1.4 2.2 4.0 5.4KC 0 1.2 1.2 1.2Polysaccharide 4.7 2.5 7.5 12.2KC 0 2.7 2.7 2.7

R. mangle (wood)Lignin 1.5 1.0 2.6 4.1Polysaccharide 4.1 2.9 8.8 12.9a KC, Killed control.

anaerobic peat from the Okefenokee Swamp, was degradedat 37% of the aerobic rate.

DISCUSSIONAlthpugh there have been previous reports of the anaero-

bic bi'degradation of lignin (2, 30), the methods used in theearlier studies relied on chemical isolation of lignin fromdecaying vegetation and gravimetric analyses for weightloss. No precise method for the quantitative chemical isola-tion of lignin from plant material exists (26), and thus, resultsfrom earlier studies were not conclusive. Radiolabeled lig-nins, either as [14C-lignin]lignocelluloses or synthetic

TABLE 2. Rates of anaerobic biodegradation of specificallyradiolabeled lignocelluloses and synthetic lignin (DHPs) expressed

as percentages of aerobic rates

% of aerobic mineralization rates

Radiolabeled substrate ["4C-ligninj lig- [14C-polysaccha-nocellulose ride]lignocellulose

LignocelluloseS. alterniflora 7.5 8.2J. roemerianus 6.0 3.8C. walteriana 33.0 40.2R. mangle (leaves) 2.9 4.9R. mangle (wood) 3.7 12.6

DHPs 1.6 (salt marsh)0.9 (Okefenokee

Swamp)

[14C]lignins, are considered to be the best substrates avail-able for determining lignin degradation rates (7). However,for degradation rates obtained with these substrates to beinterpretable, natural [14C-lignin]lignocelluloses and synthet-ic [1"C]lignins (DHPs) should be sufficiently characterizedfor the distribution of radiolabel within specific chemicalcomponents of [14C-lignin]lignocelluloses and for the molec-ular weight distribution of radiolabel in 14C-DHPs. In thisstudy, [14C-lignin]lignocelluloses, derived from a variety ofstructurally different plants, and 14C-DHPs were thoroughlycharacterized (1, 20). The presence of radiolabeled nonligninmaterial, such as protein, in [14C-lignin]lignocellulose prepa-rations can cause overestimation of lignin degradation rates(1). Benner et al. (1) found that when [14C]cinnamic acid isused as the precursor to label the lignin component ofherbaceous plants, labeling of the contaminating protein isminimized. Very low percentages (<3%) of the radiolabel inand in this study all of the [14C-lignin]lignocelluloses weredegraded to levels above the percentages of any contaminat-ing radiolabeled protein. In the radiolabeled DHPs, less than1% of the radioactivity was in low-molecular-weight (Mr <750) material (20; T. K. Kirk, personal communication).Likewise, the DHPs were degraded to levels well abovepercentages of low-molecular-weight material.Herbaceous plant lignins, especially grass lignins, contain

cinnamic acid derivatives esterified to the periphery of themacromolecule (26). These covalently bound cinnamic acidspossibly function as linkage units between core lignin andthe polysaccharides of cell walls and as precursors offurthercore lignin biosynthesis (26). Ferulic and p-coumaric acidsare the most commonly found cinnamic acids esterified tocore lignin, and they typically make up 5 to 10% of grasslignins (27). From 2.5 to 11.8% of the radioactivity in [14C-lignin]lignocelluloses derived from the herbaceous plantsused in this study is associated with cinnamic acid esters (1).None of the radiolabel from [14C-lignin]lignocellulose de-rived from mangrove wood is associated with cinnamic acidester groups (1); this is consistent with the fact that the lignincomponent of wood generally lacks ester-bound cinnamicacids (26). Benner et al. (1) presented data indicating that,under aerobic conditions, cinnamic acid esters are mineral-ized more rapidly than core lignin. Thus, it seems likely thatester-bound peripheral lignin is also more rapidly degradedthan core lignin under anaerobic conditions. Degradation ofesterified cinnamic acids may have contributed to the rela-tively faster anaerobic degradation of lignins derived fromthe herbaceous plants. However, there is no obvious rela-tionship between the anaerobic degradation rate of lignin-labeled substrates and the percentage of radiolabel associat-ed with esterified peripheral lignin. For example, 5.0% of theradiolabel in [14C-lignin]lignocellulose from J. roemerianusis associated with cinnamic acid esters (1), yet J. roemer-ianus [14C-lignin]lignocellulose was degraded (30°C) in anox-ic salt-marsh sediments more slowly than synthetic [14C]lig-nin (21 to 25°C), which contains no esterified radiolabel.There have been previous reports of the anaerobic biodeg-

radation of chemically modified 14C-labeled natural andsynthetic lignins. Colberg and Young (6) found that oligolig-nols, which were chemically solubilized from Douglas fir[14C-lignin]lignocellulose, were degraded to 14CO2 and 14CH4by a microbial consortium from a sludge digestor. Zeikus etal. (31) also used a chemically solubilized low-molecular-weight (Mr < 300) lignin derived from 14C-DHPs and foundrelatively rapid biodegradation to 14CO2 and 14CH4 in anoxicsediments from Lake Mendota (Madison, Wis.); however,biodegradation of high-molecular-weight 14C-DHPs was not

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detected (13, 31). If the biodegradation rates of high-molecu-lar-weight '4C-DHPs in anoxic Lake Mendota sedimentswere similar to the rates of degradation of 14C-DHPs whichwe detected in anoxic salt-marsh or Okefenokee Swampsediments, then the previous authors would not have beenable to detect any 14CO2 or 14CH4. The methodology weused is 10-fold more sensitive than theirs and is required todetect the very slow anaerobic rates of biodegradation of14C-DHPs. Using [14C-lignin]lignocellulose derived frompoplar wood and a methodology similar to the methodologywe used. Odier and Monties (24) did not detect anaerobicdegradation in soil enrichment cultures; however, the[14C]lignocellulose preparations were autoclaved before use.We have found that autoclaving can greatly reduce thebiodegradability of [14C-lignin]lignocelluloses (unpublisheddata).Aerobic biodegradation of the polysaccharide component

of lignocellulose is generally faster than that of the lignincomponent and appears to be tightly coupled with ligninbiodegradation (1, 7, 17, 21, 22). Similarly, under anaerobicconditions the polysaccharide components of lignocelluloseswere more biodegradable than the lignin components. Theratio of lignin to polysaccharide degraded from each ligno-cellulose was similar under aerobic and anaerobic condi-tions, suggesting a tight coupling between the anaerobicdegradation of the lignin and polysaccharide components oflignocellulose. Pure [U-14C]cellulose (ICN Radiochemicals,Irvine, Calif.) is degraded twice as fast as S. alterniflora[14C-polysaccharide]lignocellulose in anoxic salt-marsh sedi-ments (18), indicating that lignin forms a barrier to anaerobicbiodegradation of the physically and covalently bound poly-saccharides of natural lignocelluloses.

Synthetic [14C]lignin was degraded two times faster in salt-marsh sediments than in Okefenokee Swamp peat, indicatingthat anaerobic decomposition is inhibited in the low-pH peatof the Okefenokee Swamp. The relatively slower anaerobicdegradation rates of lignin in the Okefenokee Swamp maycontribute to the accumulation of plant material as peat.However, the rate of anaerobic biodegradation of C. walter-iana lignocellulose in Okefenokee Swamp peat was compa-rable to anaerobic degradation of S. alterniflora and J.roemerianus lignocelluloses in salt-marsh sediments. Thus,the accumulation of plant material as peat in the OkefenokeeSwamp is probably the result of a combination of factorsincluding not only the relatively slower degradation ratescompared with those of the salt marsh, but also the hydro-logical conditions, namely the extremely low rates of watermovement and the lack of tidal flushing.

ACKNOWLEDGMENTSWe are grateful to R. L. Crawford, D. L. Crawford, T. K. Kirk,

P. J. Colberg, L. Y. Young, J. G. Zeikus, J. K. W. Wiegel, andW. J. Weibe for advice in the preparation of this manuscript. Wealso thank W. B. Whitman for the use of his anaerobic chamber andfor advice on anaerobic procedures. Thanks also go to CaptainMorgan and the crew of O.R.V. Cape Florida.

This work was supported by National Science Foundation grantsOCE-8117834, BSR-8114823, and BSR-8215587. Additional fundingwas provided from grant NA 80AA-D00091 from National Oceanicand Atmospheric Administration Office Sea Grant, U.S. Depart-ment of Commerce.

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22. Maccubbin, A. E., and R. E. Hodson. 1980. Mineralization ofdetrital lignocelluloses by salt marsh sediment microflora. Appl.Environ. Microbiol. 40:735-740.

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27. Shimada, M., T. Fukuzuka, and T. Higuchi. 1971. Ester linkagesof p-coumaric acid in bamboo and grass lignins. Tech. Assoc.Pulp Pap. Ind. 54:72-78.

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ERRATA

Physiological Ecology of a Gliding Bacterium Containing Bacteriochlorophyll aBEVERLY K. PIERSON, STEPHEN J. GIOVANNONI, AND RICHARD W. CASTENHOLZ

Biology Department, University of Oregon, Eugene, Oregon 97403, and Biology Department, University of Puget Sound, Tacoma,Washington 98416

Volume 47, no. 3, p. 577, column 2, line 21: "Solution B consisted of sterile 0.05 M NaHCO3 (in water)" should read"Solution B consisted of sterile 0.5 M NaHCO3 (in water)."

Anaerobic Biodegradation of the Lignin and Polysaccharide Components ofLignocellulose and Synthetic Lignin by Sediment Microflora

RONALD BENNER, A. E. MACCUBBIN, AND ROBERT E. HODSON

Department of Microbiology and Institute of Ecology, University of Georgia, Athens, Georgia 30602

Volume 47, no. 5, p. 998, column 1, lines 22 and 23: . . equals aboveground production and frequently is consideredgreater . . ." should read ". . . equals aboveground production and frequently is considerably greater...."

Page 1002, column 2, lines 17-20: A portion of this sentence was inadvertently omitted. The correct sentence should read asfollows: "Very low percentages (<3%) of the radiolabel in [14C-lignin]lignocelluloses are associated with protein (1), and inthis study, all of the [14C-lignin]lignocelluloses were degraded to levels above the percentages of any contaminatingradiolabeled protein."

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