comparative pure cultures ofgreenalgae ... · compositions of algae andtheir decomposed residues...

Vol. 54, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1988, p. 1051-10600099-2240/88/041051-10$02.00/0Copyright X 1988, American Society for Microbiology

Comparative Analysis of the Chemical Composition of Mixed andPure Cultures of Green Algae and Their Decomposed Residues by

13C Nuclear Magnetic Resonance SpectroscopyJ. L. ZELIBOR, JR.,'t L. ROMANKIW,1 P. G. HATCHER,' AND R. R. COLWELL2*

U.S. Geological Survey, Reston, Virginia 22092,1 and Department of Microbiology, University of Maryland,College Park, Maryland 207422

Received 20 August 1987/Accepted 29 January 1988

It is known that macromolecular organic matter in aquatic environments, i.e., humic substances, is highlyaliphatic. These aliphatic macromolecules, predominantly paraffinic in structure, are prevalent in marine andlacustrine sediments and are believed to originate from algae or bacteria. A comparative study of mixed andpure cultures of green algae and their decomposed residues was performed by using solid-state 13C nuclearmagnetic resonance spectroscopy as the primary analytical method. Results obtained in this study confirm thepresence of components that are chemically refractory and that are defined as alghumin and hydrolyzedalghumin. These were detected in heterogeneous, homogeneous, and axenic biomasses composed of severalgenera of Chlorophyta. Although the chemical composition of algal biomass varied with culture conditions, thechemical structure of the alghumin and hydrolyzed alghumin, demonstrated by '3C nuclear magneticresonance spectroscopy appeared to be constant for members of the Chlorophyta examined in this study. Thealghumin was dominated by carbohydrate-carbon, with minor amounts of amide or carboxyl carbon andparaffinic carbon, the latter surviving strong hydrolysis by 6 N HCI (hydrolyzed alghumin). Bacterialdecomposition of heterogeneous algal biomass labeled with 13C was conducted under both aerobic andanaerobic conditions to determine chemical structure and stability of the refractory material. The refractoryfraction ranged from 33% in aerobic to 44% in anaerobic cultures. The refractory fraction recovered fromeither aerobic or anaerobic degradation comprised 40% alghumin, which represented an enrichment by 10%relative to the proportion of alghumin derived from whole cells of algae. The paraffinic component in thehydrolyzed alghumin of whole algal cells was found to be 1.8% and increased to 5.1 and 6.9% after aerobic andanaerobic bacterial degradation, respectively. It is concluded that members of the Chlorophyta contain acommon insoluble structure composed of paraffinic carbon that is resistant to chemical and bacterialdegradation under conditions used in this study. The paraffinic structure is identical to those constitutinghumin of aquatic origin. Thus, alga-derived macromolecular compounds deposited in aquatic environments(alghumin) probably contribute to sedimentary humic substances.

Decomposition is important in the recycling of elementsessential to life in the biosphere and is responsible, in part,for the deposition of residues composed of complex organicmaterial, termed humus or humic substances. Macromolec-ular organic compounds of the general group of humicsubstances have been classified on the basis of the relativesolubilities of their components. Thus, humic acids aresoluble in alkali but insoluble in acid, whereas fulvic acidsare soluble in both acid and alkali. Humic matter insoluble inboth alkali and acid is known as humin.Humic substances have been referred to as heteropoly-

condensates, implying a diverse and complex nature (32).Terrestrially derived humic substances are characterized bya predominantly aromatic chemical structure with methoxyl,hydroxyl, carboxyl, phenolic, and ether functional groups(15, 31). Lignin was thought to be a primary progenitor ofterrestrial humic materials (I. A. Breger, Ph.D. dissertation,Massachusetts Institute of Technology, Cambridge, 1950).Aquatic humic substances, in contrast, are predominantlyaliphatic (5, 6, 14, 23, 33). Recent studies have shown thatthe humin fraction isolated from aquatic sediments is com-posed of insoluble, cross-linked, macromolecular paraffinic

* Corresponding author.t Present address: Department of Microbiology, University of

Maryland, College Park, MD 20742.

structures. These structures are also present in soils andpeats (13).The origin and mechanism of accumulation of aquatic

humin were investigated in Mangrove Lake, a small salinelake in Bermuda (16). At this site, a large organic input,combined with a rapid sedimentation rate, has resulted in anaccumulation of anoxic algal sapropel having a gelatinoustexture. The organic component of the sapropel collectedfrom surface layers was observed to be dominated bycarbohydrate-carbon, with minor amounts of paraffinic car-bon. As a result of chemical and bacterial diagenesis, theparaffinic carbon component accumulated with depth anddominated the humin fraction. Results of studies based onsolid-state '3C nuclear magnetic resonance (NMR) spectros-copy and stable-isotope analytical techniques demonstratedthat the paraffinic component accumulates as a residualconcentrate; it is believed to be derived directly from micro-organisms, notably algae (16, 17). Algal populations inMangrove Lake were found to be dominated by filamentousstrains of the genus Cladophora (J. L. Zelibor, P. G.Hatcher, N. Szeverenyi, and E. G. Maciel, Northeast AlgalSymp., Woods Hole, Mass., 1983).

It is well known that genera of the family Chlorophycea-ceae are ubiquitous in aquatic environments and contain arefractory component, i.e., are resistant to bacterial decom-position under laboratory conditions for 1 year (10). The

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refractory fraction varies from 12 to 87% (mean, 40%),depending on the strain and conditions used during decom-position (10, 19, 24, 25). Thus, microorganisms appear to beprimary contributors to refractory components, but thepresence of a complex refractory paraffinic cellular structurein algae has not been conclusively demonstrated.The objectives of this study were (i) to test whether

paraffinic structures are original components of living algae,(ii) to investigate whether the paraffinic component is resis-tant to bacterial decomposition, and (iii) to compare thechemical structure of the refractory fractions from algae withthose from aquatic sediments. An investigation of the com-parative structure of that material insoluble in dilute alkaliand acid, hereafter defined as alghumin and hydrolyzedalghumin, prepared from various genera of the Chlorophytawas performed by using pure and mixed cultures. It isimportant to emphasize that alghumin was prepared by usingcultures of algae and by methods used to prepare humin fromsoils and sediments. Although the alghumin is operationallydefined here as the substance isolated by the same methodsused to isolate humin, this fraction can contain polysaccha-rides which are not humic in nature. Therefore, alghuminswere hydrolyzed by refluxing in 6 N HCI to isolate a fractionthat could be compared to humin. This fraction is referred toin this study as the hydrolyzed alghumin. Similar experi-ments using bacteria, data for which are not provided here,show the presence of bachumin, i.e., acid- and alkali-insoluble material prepared from bacterial cultures by usingthe same methods used to isolate humin.

MATERIALS AND METHODS

Algal strains used in this study. Various genera of theChlorophyta, including filamentous and unicellular strains,were collected from environments known to be depositingalga-derived humic substances (P. G. Hatcher, Ph.D. disser-tation, University of Maryland, College Park, 1980). Strainsof the genera Chlorella, Scenedesmus, Chlamydomonas,and Closterium were isolated from algal mats overlying peatdeposits in Conservation District 2, the Everglades, Fla.Strains were obtained by streak plating onto WC medium (9)amended with 15 g of agar (Difco Laboratories, Detroit,Mich.) per liter. After incubation for 5 days at 21°C underfluorescent light (14 h of light, 10 h of dark), CFU wereisolated and purified by restreaking three times, and thepurified strains were identified. Identification to the genuslevel was accomplished by determining cellular morphology(28) observed by using a compound Zeiss microscopeequipped with a x10 ocular and a x40 objective. A mixedalgal culture (MAC) was prepared by combining the fourstrains in equal proportions and growing them in liquid WCmedium. The mixed culture was examined for paraffinicstructures and also labeled with 13C for the decompositionexperiments.

Filamentous algae, composed of Zygnema sp. or Hydro-dictyon sp., were isolated from mats collected in the Okefe-nokee Swamp, Ga., and from a small pond in LoudonCounty, Va., respectively. Filamentous algae were sepa-rated from the mat by using sterile forceps under a binoculardissecting microscope (x 15), identified to the genus level bycellular morphology, and maintained in WC medium.Axenic Botryococcus braunii (Utex 572), Chlorella pyre-

nosidosa (Utex 1230), and Scenedesmus obliquus (Utex 78)were obtained from the Algal Culture Collection of theUniversity of Texas at Austin and maintained on agar slantscomposed of WC medium. Axenic Dunaliella tertiolecta, a

strain possessing only a minimal cell wall structure, i.e., therigid cell wall is not synthesized, was provided by R.Sjoblad, University of Maryland, College Park, and main-tained on agar slants containing a modified Bold's Bristolsolution (3) that was prepared by altering the concentration(grams per liter) of NaCl (12.0), CaCl2- H20 (0.15),MgSO4 7H20 (1.8), and MgCl2 * 6H2 (1.3). Also, 10 ml ofurea solution (0.4 M) and 1.0 ml of a sodium glycerophos-phate solution (0.1 M) were substituted for NaNO3,KH2PO4, and K2HPO4.

Algal mass culture. The MAC and the filamentous andaxenic strains of green algae were grown in mass culture toacquire sufficient biomass for analysis of alghumin andhydrolyzed alghumin.

Production inocula for the MAC or filamentous algalculture and axenic algal strains were prepared by transfer-ring cells to liquid (200 ml) WC medium or modified Bold'sBristol solution. Liquid cultures were incubated at roomtemperature (21 to 23°C) under fluorescent light (150 micro-einsteins per m' per s) for 14 h in the light and 10 h in thedark. Cultures were mixed by periodic shaking or continu-ous aeration, and the growth was characterized (8). Duringthe exponential phase of growth, the production inoculumwas added to the appropriate production medium (12 liters)prepared in 18-liter sterile Pyrex carboys (Fisher ScientificCo., Pittsburgh, Pa.). The apparatus used was similar to thatdesigned for mass growth of axenic algal cultures (8). Thealgal mass cultures were incubated at room temperature (21to 23°C) under fluorescent light (150 microeinsteins per M2per s) for 14 h in the light and 10 h in the dark.The axenic algal biomass was tested periodically for the

presence of bacteria by using both culture and microscopemethods. Samples of the algal culture were streaked (threetimes) onto nutrient agar (Difco), plate count agar (Difco),triple-sugar agar (Difco), and Tween 80 hydrolysis agar. TheTween 80 hydrolysis agar was composed of (grams per liter)peptone (10.0), NaCl (5.0), CaCI2- H20 (0.1), Tween 80(polyethylene sorbitan mono-oleate) (10.0 ml), and agar(15.0). All ingredients except Tween 80 were dissolved inwater, sterilized by autoclaving, and cooled to 50°C. Tween80 was filter sterilized (0.2-,um pore size; Nuclepore Corp.,Pleasanton, Calif.) and added aseptically before the plateswere poured. All plates were incubated for several days at21°C, after which they were examined for the presence ofbacterial colonies. Bacterial colonies showing a clear zonewere recorded as positive for lipid hydrolysis. In addition tobeing cultured, samples of algae were stained with a solutionof crystal violet (1.0% [wt/vol]) and examined for the pres-ence of bacteria by using a compound light microscope(Zeiss Photomicroscope III) equipped with a x 10 ocular anda x 150 oil immersion objective. In some cases, initialattempts to grow axenic algae in mass culture did not yieldbiomass free of bacteria. However, after several attempts,axenic mass culture was achieved without antibiotics in theculture medium.

Algal cells in mass culture were harvested during expo-nential growth by using a Sorvall RC2-B centrifuge equippedwith a KSB continuous flow system (Ivan Sorvall, Inc.,Norwalk, Conn.). Cells were washed several times withsterile distilled water and lyophilized.

Incorporation of stable isotopes into algal cells in MAC.Stable isotope ('3C) was incorporated into the MAC toprovide higher sensitivity for solid-state 13C NMR analysis.The MAC culture, comprising strains of Chlamydomonas,Chlorella, Closterium, and Scenedesmus, was used for la-beling cultures with the stable isotope and for subsequent

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COMPOSITIONS OF ALGAE AND THEIR DECOMPOSED RESIDUES

FIG. 1. Closed batch system used to culture algal biomass en-

riched in stable isotope (13C).

bacterial decomposition studies. The genera of Chlorophytaselected for study are ubiquitous in freshwater aquaticenvironments and have been shown to exhibit a slight tomoderate resistance to bacterial decomposition (10). Cultureconditions for labeling MAC with 13C were modified fromthose described above by using a closed batch system. Theculture was mixed by gassing with air enriched by 13CO2.The 13C-labeled MAC culture was grown in WC medium

containing 13CO2. Inocula for these strains were prepared as

described above and added to 12 liters of medium in an

18-liter Pyrex carboy. The culture was sealed with a rubberstopper fitted with glass tubing and connected to a dia-phragm pump by using Tygon tubing (Sani-Tech) (Fig. 1). Anadjustable clamp was used to regulate gas flow for mixing theculture. Erlenmeyer flasks (250 ml) with rubber stoppersfitted with glass tubing served as water traps. C02-enrichedby 13C (10 to 20% [vol/vol]) was added periodically (seeTable 4). To do this without dilution with atmospheric C02,a 150-ml sidearm test tube fitted with a stopper was con-

nected to the tubing on the outlet side of the pump. Anadjustable clamp was used to close the line, while a mixtureof NaHCO3 (1.5 g) and NaH13C03 (0.05 g) (90 atom% 13C;Merck Sharp & Dohme Canada Ltd., Montreal) was addedto the test tube. The side arm of the test tube was carefullyfilled with concentrated H3PO4, the tube was fitted in place,and the adjustable clamp was opened. Carbon dioxide gaswas generated by mixing the acid with the bicarbonatemixture and feeding the mixture into the line by the resultingpositive pressure. An 18-liter carboy served as a gas-mixingchamber before the gas was bubbled through the productionmedium.

Indicators of algal growth, such as growth rate, dissolvedoxygen, nitrate concentration, and pH, were monitoredthroughout the incubation period. Algal growth rate wasmonitored by measuring A750. Dissolved oxygen was mea-sured by using an oxygen meter (Orbsphere model 2607)equipped with an oxygen probe. The pH of the medium wasmeasured with a model 130 pH meter (Corning Glass Works,Corning, N.Y.) equipped with a semimicro combinationelectrode. Nitrate concentration was determined in the algalculture medium by using a Dionex model 14 ion chromato-graph.

At the end of the incubation period (12.7 days), cells wereharvested by using a Sorvall RC2-B centrifuge equipped witha KSB continuous flow system, washed three times with

sterile distilled water, and lyophilized. This labeled algalbiomass was used in the in vitro aerobic and anaerobicdecomposition experiments. 13C content in CO2 from com-busted labeled algal biomass was determined by using ahand-crafted, high-precision isotope ratio mass spectrometerand standard methods (7; E. C. Spiker, unpublished data).Samples were combusted in sealed quartz tubes and dilutedwith unlabeled CO2 prior to analysis (34).

In vitro bacterial decomposition of algal biomass labeledwith stable isotopes. The lyophilized MAC biomass enrichedwith "3C was decomposed under aerobic or anaerobic con-ditions, and the residual particulate matter was collected foranalysis. The apparatus used to carry out the two decompo-sition experiments was similar to that used by others (24, 25)to study the production of dissolved organic matter fromgreen algal cells subjected to bacterial decomposition.Each of two 4-liter glass bottles was filled with 3 liters of

a medium composed of KH2PO4 (33.0 mM) and Na2HPO4(33.0 mM) in distilled water. The pH of the medium wasadjusted to 7.0 by using 2 N H3PO4. The bottles containingthe medium were placed in a water bath maintained at 30°Cin the dark. The bottle to be used for the aerobic decompo-sition was gassed for 48 h with zero-grade air. The anaerobicbottle was purged for 48 h with nitrogen made oxygen free bybeing passed over copper shavings heated to 300°C (BellcoGlass, Inc., Vineland, N.J.). Following gas purges, 6.5 g oflyophilized labeled MAC biomass was added to each bottleunder a positive gas pressure. At the same time, the bottleswere inoculated with 50.0 ml of a bacterial suspensionprepared by mixing 10 g of peat, previously collected fromthe Tamiami Trail, the Everglades, Fla., to dilution blankscomposed of 90.0 ml of phosphate buffer (33.0 mM KH2PO4and 33.0 mM Na2HPO4 in distilled water). Prior to theaddition of the peat, dilution blanks were made eitheraerobic by aeration with sterile ultrapure air or anaerobic bythe addition of sodium thioglycolate (0.02%) and cysteinehydrochloride (0.01%) and gassing with oxygen-free nitro-gen. Bacto-Reasurin (2.0 mg; Difco) was added as an indi-cator of anaerobiosis. The suspended peat samples weresealed with rubber stoppers and vigorously shaken, and themixture was centrifuged (1,024 x g for 1.5 min at 21°C). A50-ml sample of either the aerobic or anaerobic supernatantwas added to the appropriate bottle containing the labeledalgae. The resulting cultures were incubated for 157 days at30°C and mixed by bubbling with the appropriate gas.To monitor the progress of bacterial decomposition, sub-

samples were collected periodically and the numbers ofviable proteolytic, lipolytic, and cellulolytic bacteria weredetermined. Total viable numbers of aerobic and anaerobicbacteria were determined by using nutrient agar (proteo-lytic), Tween 80 hydrolysis agar (lipolytic), and celluloseagar (cellulolytic) (20). Cellulose agar (18), used for aerobicbacterial enumeration, was prepared without a reducingagent. Colonies showing a clear zone were recorded aspositive for cellulose degradation. Aliquots of samples (1.0ml) from either the aerobic or anaerobic decompositionsystem were serially diluted in appropriate dilution blanks(9.0 ml), and 0.1 ml of the dilution was spread plated.Anaerobic serial dilutions were performed by using the VPIanaerobic transfer system (Bellco) and prereduced dilutionblanks composed of phosphate buffer. Media used to growanaerobes were reduced by using the method to preparereduced phosphate buffer. Reduced media were spreadplated in a nitrogen-filled glove bag. Plates were incubated at21°C either under aerobic conditions or in a nitrogen atmo-sphere in an anaerobic incubator. After 5 days, plates

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containing between 30 and 300 CFU were counted. Theplates were incubated for an additional 5 days and countedagain. The decompositions were terminated when the viablebacteria in each physiological group was no longer in astationary phase of growth.The rates of decomposition for the aerobic and anaerobic

decompositions were calculated based on the concentrationof residual particulate matter at different times. Particulatematter was collected in triplicate at each time interval (seeTable 6) by centrifugation (12,000 x g for 10 min at 4°C),followed by washing (three times) and lyophilization. Theconcentration of residual particulate matter was determinedby weighing residues collected from 20-ml subsamples.Upon termination of in vitro decompositions, residual

particulate matter (decomposed residue) was collected bycentrifugation (29,000 x g for 10 min at 4°C), washed threeor more times with distilled water, and lyophilized. LabeledMAC biomass and the decomposed residues were analyzedfor ash content, elemental carbon, hydrogen, and nitrogen,and percent (wt/wt) alghumin.

Isolation of insoluble residues (alghumin). Insoluble resi-dues were isolated from MAC, Zygnema sp., Hydrodictyonsp., axenic strains, labeled MAC, and the decomposedlabeled MAC particulate residues by using methods similarto those for extracting humic substances from sediments andsoils (30). Gram quantities (1 to 3 g) of lyophilized samplewere extracted by Soxhlet (13) by using a 1:1 (vol/vol)mixture of benzene:methanol to remove lipids. Soxhletextraction was followed by treatment with 0.01 N HCl toremove low-molecular-weight organic compounds. Humicmaterials were extracted by subsequent treatment with 0.5 NNaOH. Soluble substances (humiclike and fulviclike acids)were separated from the insoluble fraction (alghumin) bycentrifugation. Alghumin fractions were washed severaltimes with distilled water and acidified to pH 6.0 by theaddition of HCI, followed by repeated washes. Some of thealghumin was lyophilized and stored for analysis. Most ofthe alghumin was treated with sodium paraperiodate toremove carbohydrates, as described by Ritchie and Purves(29), and then with 6 N HCl under reflux for 2 h to hydrolyzeproteinaceous materials. The residue from such a treatmentwas called hydrolyzed alghumin.

Chemical and spectroscopic methods. Solid-state "3C NMRanalyses were performed by using the cross-polarization,magic-angle spinning (CPMAS) technique. Spectra wereobtained on either a spectrometer constructed in the labora-tory at Colorado State University or a Chemagnetics (FortCollins, Colo.) 100S/200L NMR spectrometer operating at afield strength of 2.35 T ('H field of 100 MHz). ConventionalCPMAS spectra were obtained by using a 1-ms contact time,a pulse delay of 0.7 to 1 s, and a sweep width of 10 kHz. Thechemical shifts were referenced to tetramethylsilane, and anexponential filter, equivalent to 40 Hz of line broadening,was applied. Details of the method have been publishedelsewhere (12, 13). Chemical shifts are reported as parts permillion downfield of tetramethylsilane. Peak areas weredetermined by integrating NMR spectra with a Numonics(Lansdale, Pa.) digitizer (model 253) and associated tochemical functional groups (1).Ash content of the samples was determined by dry weight

difference, after the samples were heated in tared cruciblesovernight at 750°C.

Elemental analyses (C, H, N, and 0) were obtaineddirectly by using a Perkin-Elmer 240B CHN analyzer (ThePerkin-Elmer Corp., Norwalk, Conn.) or a Carlo Erba model1106 elemental analyzer.

TABLE 1. Algal strains used in this study and theirhumin isolate composition

Type and source Algal strain Alghumin(wt/wt)

Algae in nonaxenic cultureConservation District 2, MAC (Chlorella sp., 29.9The Everglades, Fla. Chlamydomonas

sp., Closterium sp.,Scenedesmus sp.)

Prairie-Folkson, Okefeno- Zygnema sp. 38.8kee Swamp, Ga.

Loudon County, Fairfax, Hydrodictyon sp. 35.7Va.

Axenic algaeUniversity of Texas Algal Botryococcus braunii 27.9

Culture Collection Chlorella pyrenosidosa 40.2Scenedesmus obliquus 48.2

University of Maryland, Dunaliella tertiolecta 2.4R. Sjoblad (no cell wall)

RESULTS

Algal communities were examined to identify genera po-tentially contributing to the macromolecular paraffinic struc-tures identified in aquatic humin. Microscope examination ofsamples of the naturally occurring algal communities col-lected from a site in the Okefenokee Swamp, Ga., showedthat members of the Chlorophyta were composed mainly ofa few genera of filamentous algae, including Desmidium,Hydrodictyon, and Zygnema. Examination of algal commu-nities inhabiting sites in the Everglades, Fla., revealed awide variety of algal types, including species of Gompho-sphaeria, Anacystis, Oscillatoria, Chlorella, Chlamydomo-nas, Closterium, Palmella, Pediastrum, Scenedesmus, Na-vicula, Diatoma, and Cyclotella. The mat was dominated byunicellular genera of the Chlorophyta. The algal mat col-lected from a small farm pond in Loudon, Va., was com-posed almost entirely of Hydrodictyon spp. NonaxenicMAC, composed of strains of Chlorella, Chlamydomonas,Closterium, and Scenedesmus, nonaxenic filamentous algae,Zygnema sp., and Hydrodycton sp. were grown underlaboratory conditions and analyzed for the presence andyield of alghumin. Also, axenic B. braunii, C. pyrenosidosa,S. obliquus, and D. tertiolecta were similarly examined.The percentage of the alghumin extracted varied among

the unicellular and filamentous green algae (Table 1). Thepercent ranged from 2.4% (wt/wt) for axenic D. tertiolecta(strain with a minimal cell wall) to 48.2% (wt/wt) for S.obliquus. The "3C NMR spectra of the nonlabeled MACculture (with associated bacteria) from the Everglades, Fla.,and the alghumin from this culture are shown in Fig. 2. Thespectrum of the whole cells was dominated by peaks at 50 to105 ppm, related to ethers and carbohydrates or polysaccha-rides. This finding was not surprising, considering thatinsoluble carbohydrates (cellulose) are known to be majorcomponents of green algal cell walls (21). Other peaks in thespectrum of the whole cells were identified as carboxyl oramide carbons (160 to 190 ppm), aromatic or olefinic carbons(110 to 160 ppm), and paraffinic carbons (0 to 50 ppm). Lipidcarbon and protein carbon are most likely responsible for theintensities in these regions of the spectrum. A spectrum ofthe alghumin obtained from the nonlabeled MAC culture isalso shown in Fig. 2. Prominent peaks are those of carbo-hydrate (50 to 105 ppm), carboxyl and amide (160 to 190ppm) carbons, and paraffinic (0 to 50 ppm) carbon.



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TABLE 2. Areas (percent) of peaks located in the variousregions of the CPMAS 13C NMR spectra of alghumin

from mixed and axenic algal biomasses

% of peaks located in area (ppm)a:Strain

0-50 50-105 110-160 160-190

MAC (Chlorella sp., 12.4 62.9 8.1 9.7Chlamydomonassp., Closterium sp.,Scenedesmus sp.)

Zygnema sp. 16.5 50.4 23.2 8.2Botryococcus braunii 18.8 42.3 11.5 11.0Chlorella pyrenosidosa 20.3 62.9 4.3 13.9Scenedesmus obliquus 26.3 59.0 4.2 10.7Dunaliella tertiolecta 30.0 44.1 3.7 22.4

a Chemical shift measured in parts per million relative to tetramethylsilane.

200 100 0 ppm

FIG. 2. CPMAS 13C NMR spectraalghumin from the Everglades, Fla.

of nonlabeled MAC and

Similar observations were made from NMR spectra ofwhole cells, alghumin, and hydrolyzed alghumin from Zyg-nema sp. and associated bacteria, isolated from the Okefe-nokee Swamp, Ga. (Fig. 3). The spectrum of whole cells ofZygnema sp. was almost identical to that of the MACculture. After strong hydrolysis (6.0 N HCI), the NMRspectrum of the hydrolyzed alghumin showed a loss ofcarbohydrate-carbon (50 to 105 ppm) and carboxyl or amidecarbon (160 to 190 ppm), with a subsequent increase inrelative percent paraffinic carbon.The relative areas (percent) of the major peaks in the

regions from 0 to 50, 50 to 105, 110 to 160, and 160 to 190ppm were calculated by integration for the alghumin of thealgal cultures used in this study (Table 2). Aliphatic carbonconstituted 12.5% (wt/wt) of the alghumin from the nonla-beled MAC culture and 16.5% (wt/wt) of the alghumin fromthe Zygnema sp. These results are consistent with thosereported for filamentous strains of raw Hydrodictyon(Hatcher, Ph.D. thesis). The relative areas (percent) ofaliphatic carbon values calculated for the axenic strains usedin this study were larger than those for the nonaxenic MAC

Whole cells

Mr/~'

200 100 0 ppm

FIG. 3. CPMAS 13C NMR spectra of nonlabeled whole cells,alghumin, and hydrolyzed alghumin in Zygnema sp. from Okefeno-kee Swamp, Ga.

and the Zygnema culture. The values were 30.0% (D.tertiolecta), 26.3% (S. obliquus), 20.3% (C. pyrenosidosa),and 18.8% (B. braunii). These data suggest that selectedgenera of green algae (with and without associated bacteria)contain insoluble residues (alghumin) exhibiting a structuredominated by carbohydrate-carbon and paraffinic carbon.The structure of the hydrolyzed alghumin for each strain iscomposed solely of paraffinic structures. Moreover, thestructures of the alghumins and hydrolyzed alghumins, asshown by NMR spectroscopy, are consistent for severalgenera, the latter resembling humin deposited in aquaticsedimentary environments (13).

Results obtained from NMR analysis of axenic whole cellsof B. braunii, D. tertiolecta, C. pyrenosidosa, and S. obli-quus, their alghumins, and hydrolyzed alghumins are shownin Fig. 4. The spectra of the whole cells varied dramatically,most likely a function of the growth conditions used. Spectrawere dominated by carbohydrate-carbon, aliphatic carbon,and amide or carboxyl carbon. The results of elementalanalyses of the algal whole cells, alghumin, and hydrolyzedalghumin for each axenic strain are shown in Table 3. TheC/N ratios of the whole cells varied from 4.7 (D. tertiolectaand B. braunii) to 10.3 (C. pyrenosidosa and S. obliquus).The C/N ratios increased following chemical treatment toisolate the hydrolyzed alghumin fraction, likely the result ofthe loss of proteins. The H/C ratios were 1.6 to 1.7 for thestrains studied. The NMR spectra of the alghumin for all thestrains were found to be comparable. The chemical structureof the alghumin was dominated by carbohydrate-carbon,with relatively minor amounts of aliphatic carbon and amideor carboxyl carbon. Acid hydrolysis resulted in the loss ofcarbohydrate-carbon and amide or carboxyl carbon, leavinga structure composed almost exclusively of paraffinic car-bon. This structure contained minor amounts of nitrogen andoxygen that varied depending on the strain examined (Table3). The NMR spectra of the hydrolyzed alghumin from theaxenic strains all appear to show a similar dominance ofparaffinic carbon. The H/C ratios varied slightly among thehydrolyzed alghumins from the axenic strains and rangedfrom 1.7 (C. pyrenosidosa) to 1.8 (D. tertiolecta). Aquatichumin composed of paraffinic carbon has a H/C ratio of 1.5(16).

Interestingly, the yield of alghumin isolated from D.tertiolecta, a mutant strain lacking most of its cell wall, wassignificantly lower (2.5% [wt/wt]) than the yields from theother types studied (27.9 to 48.2%). The NMR spectrumprepared for whole cells of this strain was similar to thatobtained for B. braunii, except that peaks appearing in the

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Whole cells Alghumin Hydrolyzed Alghumin

Chlorella pyrenosidosa

Scenedesmus obliquus

Botryococcus braunii

Dunaliella tertiolecta

. . 1

200 100 0 ppm 200 100 0 ppm

FIG. 4. CPMAS "3C NMR spectra of whole cells, alghumin, and hydrolyzed alghumin in the axenic species C. pyrenosidosa, S. obliquus,B. braunii, and D. tertiolecta.

regions of 50 and 105 ppm were reduced, an observationconsistent with the suggestion that cell wall structures con-tribute to sedimentary humic structures (26). Recent studies(2) of cell walls of B. braunii indicate the presence of abiopolymer accounting for 9% (wt/wt) of the cell dry weightand resistant to chemical degradation.

Culture conditions for incorporation of '3C into the MACyielded ca. 3 to 4 g (lyophilized weight) per 12 liters ofculture, determined by using a closed system for batch

TABLE 3. Elemental analysis of whole cells, alghumins, andhydrolyzed alghumins of axenic algae used in this study

Sample % C % H % N % O H/C C/N

Dunaliella tertiolecta 50.8 6.6 12.7 29.9 1.6 4.7Alghumin 45.7 7.3 9.3 37.7 1.9 5.8Hydrolyzed alghumin 66.6 9.9 7.1 16.4 1.8 10.9

Chlorella pyrenosidosa 50.7 7.0 5.7 36.6 1.7 10.3Alghumin 47.7 6.9 6.8 38.6 1.7 8.2Hydrolyzed alghumin 71.5 10.0 3.4 15.0 1.7 24.6

Scenedesmlus obliquus 52.6 7.2 5.9 34.3 1.6 10.3Alghumin 52.0 7.6 5.8 34.6 1.7 10.4Hydrolyzed alghumin 74.2 10.8 1.8 13.7 1.8 48.5

Botnyococcus br-autnii 61.7 8.6 4.9 24.9 1.7 4.7Alghumin 47.0 6.9 7.1 39.0 1.8 7.8Hydrolyzed alghumin 76.5 10.9 1.2 11.4 1.7 73.2

culture. '3C (15 to 20%) was successfully incorporated intoalgal biomass by using the method described above. Alka-linity of the WC medium was successfully controlled byperiodic addition of CO2 (Table 4). Substitution of Na'3CO3or NaH13CO3 for '3C02 did not yield labeled biomass,indicating that 13C02 was used as the carbon source by thesealgae. Although dissolved oxygen reached saturated levelsduring the incubation period, nitrate concentration (<1p.g/ml) apparently limited exponential-phase growth after

TABLE 4. Measured parameters of '3C-labeled MAC grownin a batch closed system

Time pH Dissolved DO" A750 [NO3] C02*(day) pH 0 A5 (p.g/ml)

0 6.88 6.18 9.50 0.012 78.0 +0.7 6.78 8.95 9.20 0.024 ND"2.0 6.76 6.59 9.20 0.024 ND +3.0 6.75 7.47 9.20 0.060 71.33.7 6.76 8.37 9.20 0.074 ND +5.0 6.70 ND ND ND ND +7.7 6.87 9.58 9.20 0.236 39.3 +9.8 6.84 14.20 10.20 0.313 20.9 +

10.8 6.97 13.19 9.00 0.368 <1.0 (78.0)"12.0 8.21 9.93 9.30 0.578 ND +12.7 7.23 9.41 9.20 0.574 ND

"Dissolved oxygen (DO) measured at saturation.7 + Addition of 5% (vol/vol) CO2 enriched with 13C (20% [wt/wt]).ND, Not determined.

"NO3 (78.0 ,g/mI) added after 10.8 days.

200 100 0 ppm



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3 C labelled MAC

a)Whole

b)Alghumin

Mangrove Lake

d)Whole sapropel

e)

Huminj

Hydrolyzed Alghumin

200 100 0 ppm 200 100 0 ppm

FIG. 5. CPMAS 13C NMR spectra of "3C-labeled MAC (a),alghumin from labeled MAC (b), hydrolyzed alghumin from labeledMAC (c), sapropel from Mangrove Lake, Bermuda (d) (afterHatcher [12]) humin isolated from Mangrove Lake sapropel at a

depth of 9 m (e) (after Hatcher [16]).

incubation for ca. 11 days. The addition of nitrate (78 ,ug/ml)stimulated algal growth. Periodic microscopic examinationof the 13C-labeled MAC showed that the relative proportionof each strain in the mixed population remained constantduring the incubation period.The '3C NMR spectra of the labeled MAC whole cells,

alghumin, and hydrolyzed alghumin are shown in Fig. 5. The'3C NMR spectrum of labeled MAC whole cells is dominatedby peaks at 0 to 50 ppm and 110 to 160 ppm, likely associatedwith lipid carbon and protein carbon (Fig. 5a). The lipidcontent of labeled MAC was 32.6% (wt/wt), calculated byweight difference after extraction of the biomass with sol-vent. The spectrum for the alghumin of the 13C-labeled MAC(Fig. Sb) was comparable with the spectrum of that for thelabeled whole cells. Areas calculated from NMR spectra for'3C-labeled MAC were larger by 26% for the aliphatic region(O to 50 ppm), by 47% for the aromatic or alkene region (110to 160 ppm), and by 25% for the carboxyl or amide region(160 to 190 ppm) compared with values for nonlabeled MAC.Acid hydrolysis of '3C-labeled MAC alghumin with 6 N HCIresulted in loss of carbohydrate-carbon (50 to 105 ppm) andcarboxyl or amide carbon (160 to 190 ppm), with a subse-quent relative increase in paraffinic carbon (0 to 50 ppm)(Fig. 5c).

During in vitro aerobic and anaerobic decomposition of13C-labeled MAC, the numbers of viable cellulolytic, lipo-lytic, and proteolytic bacteria were monitored. During thefirst 42 days, the numbers of bacteria in each physiologicalgroup increased, followed by a gradual decrease (Table 5).Throughout the decomposition experiments, the numbers ofproteolytic bacteria were greater than the numbers of lipo-lytic bacteria in both aerobic and anaerobic systems. Lipo-lytic bacteria occurred in greater numbers than did cellulo-lytic bacteria. In general, anaerobes were present in fewernumbers than were aerobes. Subsamples collected from bothsystems after 157 days of incubation showed no viable (<200CFU/ml) cellulolytic bacteria. Therefore, the decomposi-tions were terminated at this time.To evaluate the progress of bacterial decomposition, par-

ticulate matter concentration, as well as the elemental com-

position of the particulates, was monitored. Although largepopulations of bacteria were present throughout decompo-

TABLE 5. Numbers of viable bacteria enumerated duringin vitro decomposition of the labeled MAC

No. (log CFU/ml) of viable bacteria

System Time (day) of on type of agar:decomposition Tween 80

Cellulose Nutrient hydrolysisAerobic 0 <2.30 6.18 3.41Anaerobic 0 <2.30 5.90 4.87

Aerobic 10 <2.30 7.18 4.40Anaerobic 10 3.28 6.60 5.20

Aerobic 24 3.48 7.80 6.70Anaerobic 24 3.58 7.40 5.62




Aerobic 157 <2.30 6.93 4.77Anaerobic 157 <2.30 4.86 4.90

sition, the percentage of MAC biomass degraded remainedunchanged after 24 days (Table 6). Slightly more biomasswas decomposed aerobically (67% [wt/wt]) than anaerobi-cally (56 [wt/wt]). The rate of decomposition, indicated bythe loss of particulate matter, was 60.8 ,ug per ml per dayunder aerobic decomposition and 50.0 ,ug per ml per dayanaerobically after 24 days. The rate of decompositionslowed by half after 42 days and to less than 10 ,ug per ml perday at the termination of the experiment at 157 days.Particulate matter showed a slightly larger C/N ratio relativeto that of the lyophilized algae (Table 7), suggesting a loss ofnitrogen-containing compounds, i.e., proteins. The particu-late fraction from the aerobic system was composed ofstructures with a lower C/N ratio compared with the fractionfrom the anaerobic system. Percent ash, carbon, hydrogen,and nitrogen were comparable in residues of both systems.The total residual particulate matter recovered after the

'3C-labeled MAC biomass was decomposed measured 32.8%(wt/wt) for the aerobic system and 43.7% (wt/wt) for theanaerobic system. The alghumin in the recovered particu-

TABLE 6. Rates of in vitro bacterial decompositionof '3C-labeled MAC

Rate ofTime Particulate Particulates decompositionSystem (day) concn recovered prgday

(mg/ml) ~~~per day)

Nondecomposed MAC 0 2.17

Decomposed MACAerobic 24 0.71 32.7 60.8Anaerobic 24 0.97 44.9 50.0



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TABLE 7. Elemental analysis of residual particulate matter atselected times during in vitro bacterial decomposition

of "C-labeled MAC

System Time % Ash % C % H % N C/N(day)

Nondecomposed MAC 0 1.0 43.2 6.1 7.1 6.9

Decomposed MACAerobic 24 1.0 47.1 6.7 8.4 6.2Anaerobic 24 1.0 48.9 6.8 7.8 7.0

Aerobic 42 1.0 47.5 6.9 8.4 6.3Anaerobic 42 1.0 48.4 6.9 7.7 7.1

Aerobic 157 3.1 48.7 7.0 8.7 8.1Anaerobic 157 3.3 50.1 7.2 8.2 9.6

Decomposed MACalghumin

Aerobic 157 48.8 7.8 8.9 6.4Anaerobic 157 51.1 8.2 9.8 6.1

Decomposed MAC hydro-lyzed alghumin

Aerobic 157 71.9 10.4 3.7 22.5Anaerobic 157 73.5 10.0 4.0 21.4

lates increased from 29% (wt/wt) in the "C-labeled MACwhole cells to 43% (wt/wt) in both the aerobic and anaerobicsystems (Table 8). Also, the hydrolyzed alghumin increasedfrom 1.8% (wt/wt) in the labeled MAC to 5.1% (wt/wt) in theaerobic and 6.9% (wt/wt) in the anaerobic system. Theelemental compositions of the alghumins and hydrolyzedalghumins were similar for the aerobic and anaerobic decom-positions (Table 7). Thus, bacterial decomposition of thelabeled MAC under the conditions described resulted in arelative increase in the alghumin and hydrolyzed alghuminfractions.The alghumins and hydrolyzed alghumins for the recov-

ered particulates from the aerobic and anaerobic systemswere analyzed by using solid-state 13C NMR spectroscopy.The spectra obtained (Fig. 6) are comparable with those ofthe undecomposed "C-labeled MAC (Fig. Sa, b, and c).Decomposed residues generated under anaerobic conditionsshowed slightly greater loss of carbohydrate-carbon andcarboxyl or amide carbon relative to those generated underaerobic conditions. This may be owing to the observationthat fewer intact cells were found during anaerobic thanduring aerobic decomposition. Results of aerobic decompo-sition, as observed by NMR analysis, were similar to resultsfor the anaerobic system, resulting in a slight loss of aliphaticcarbon (6 to 8%), carbohydrate-carbon (6 to 8%), andaromatic or alkene carbon (2 to 4%) and in a relatively larger

TABLE 8. Percent alghumin and hydrolyzed alghumin in residualparticulate matter from in vitro bacterial decomposition

of "C-labeled MACTotal residual . Hydrolyzed

System particulates Alghumn aghumin

Nondecomposed MAC 27.9 1.8

Decomposed MACAerobic 32.8 39.9 5.1Anaerobic 43.7 39.7 6.9

Aerobic

Residual particulate matter

Alghumin

Hydr

200 100 0 ppm

rolyzed A1g

Anaerobic

ghumin

200 100 0 ppm

FIG. 6. CPMAS 13C NMR spectra of various fractions obtainedin the "C-labeled MAC aerobic and anaerobic bacterial decompo-sitions.

loss of amide or carboxyl carbon (24 to 26%). The NMRspectra of the alghumins recovered from the particulatefraction of the decomposed algae were nearly identical to thespectra of those from the particulate fraction. The hydro-lyzed alghumins from both the aerobic and anaerobic decom-positions were composed entirely of paraffinic carbon (0 to50 ppm).The areas (percentage) of the major peaks in the regions

from 0 to 50, 50 to 105, 110 to 160, 160 to 190 ppm for thelabeled MAC, decomposed particulates, alghumins, andhydrolyzed alghumins were calculated (data not shown).Decomposition of the labeled MAC resulted in a slightdecrease in aliphatic carbon, carbohydrate-carbon, and aro-matic carbon in the residual particulates for both the aerobicand anaerobic systems. The C/N ratio for the decomposedlabeled MAC increased from 6.9 to 8.1 (aerobic) and 9.6(anaerobic), likely due to the decomposition of algal proteinsand lipids. The alghumins from the residual particulates weresimilar in structure to those from the residual particulatesfrom both aerobic and anaerobic systems. The hydrolyzedalghumins showed a relative increase in paraffinic carbon,with subsequent decreases in carbohydrate-carbon, aro-matic carbon, and carboxyl or amide carbon.

DISCUSSIONA comparative analysis of the chemical structures of

mixed and pure cultures of green algae and their decom-posed residues provided information about recalcitrant mac-romolecular structures derived from green algae. Thesestructures probably are deposited throughout various phasediscontinuities in sediments and play a role in geochemicalprocesses, e.g., formation of humic substances.

Several studies investigating the rates and processes in-volved in the decomposition of various eucaryotic algalbiomasses (10, 19, 22, 24, 25) have been done. However,results reported in this study are based on a nondestructiveanalytical method that yields structural information. Thepresence of chemically refractory components (alghuminand hydrolyzed alghumin) in raw heterogeneous, homoge-neous, and axenic biomasses composed of genera in theChlorophyta has been confirmed. Although the chemicalcomposition of algal biomass can vary with culture condi-tions, the chemical structure of hydrolyzed alghumin (as



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shown by 13C NMR spectroscopy) appears to be constantwithin the members of the Chlorophyta that were studied.The alghumins were dominated by various amounts ofcarbohydrate-carbon, with minor amounts of aliphatic car-bon (paraffinic), the latter surviving strong hydrolysis by 6 NHCI. The hydrolyzed alghumins prepared from axenic algalbiomass were composed almost exclusively of paraffiniccarbon.

Bacterial decomposition of heterogeneous algal biomasslabeled with "3C was conducted under both aerobic andanaerobic conditions to investigate the chemical structureand stability of the refractory material. Incorporation of 13Cin algal biomass facilitated solid-state "3C NMR analysis bydecreasing the analysis time and providing spectra of betterresolution than those for unlabeled algal biomass. The het-erogeneous algal populations were observed to decomposeslowly and incompletely when inoculated with cultures ofheterotrophic bacteria. The algae were found to vary insusceptibility to decomposition according to genus. Therefractory fraction (residual particulate matter) ranged from33% in the aerobic system to 44% in the anaerobic system.These values are similar to those reported for bacteriallydegraded green algal biomass (40% aerobic, 44% anaerobic)(19). The residual particulate matter recovered from theaerobic and anaerobic decompositions was composed ofabout 40% (wt/wt) alghumin (10% enrichment, i.e., greaterrelative to algal whole-cell preparations). The hydrolyzedalghumin (paraffinic carbon) composed 1.8% of the 13C-labeled algae and increased after bacterial decomposition to5.1% (aerobic) versus 6.9% (anaerobic). Thus, it is con-cluded that macromolecular paraffinic structures are, in-deed, components of living algal biomass. Also, the paraf-finic structures are resistant to bacterial and chemicaldecomposition.A good deal of speculation has been offered concerning

the origin of aquatic humic substances and the mechanism(s)involved in their formation. Several hypotheses have beenproposed to explain the origin of the aliphatic structures,including condensation of simple biomolecules, e.g., carbo-hydrates and proteins (23, 35), postmortem condensation oflipids (4), and condensation of decomposition products withcomponents of cell wall residues of microorganisms (26, 27).A more recent hypothesis (16) proposes that humic sub-stances are preformed macromolecular algal and/or bacterialcomponents selectively preserved during early diagenesis.A striking similarity was noted when solid-state '3C NMR

spectra of labeled MAC whole cells were compared withthose of sapropel at surface intervals of sediments in Man-grove Lake, Bermuda, a marine ecosystem in which filamen-tous Cladophora sp. is a major contributor to the depositedorganic matter (13) (Fig. 5a and d). Hydrolyzed alghumin,prepared from '3C-labeled MAC and composed primarily ofparaffinic carbon, was almost identical to the humin isolatedfrom sapropel at a depth of 9 m (Fig. 5c and e). Thus, theresults of this study support the hypothesis of selectivepreservation (16), evidenced by the similarity in amount andstructure of alghumins and hydrolyzed alghumins producedby algae with humin from aquatic sediments.We suggest that preformed algal cell wall components are

the primary source of alghumin. Convincing evidence isprovided by the observation that the algal strain containing aminimal cell wall was nearly devoid of alghumin. Further-more, it is hypothesized that a possible mechanism forresistance of carbohydrate-carbon in algal cell walls tochemical and bacterial decomposition is the association withminor amounts of paraffinic carbon rather than polyaromatic

compounds (10). Association of cell wall carbohydrates(cellulose strands) with a macromolecular paraffinic compo-nent may provide protection against extracellular bacterialcellulase by altering cellulose conformation and therebyreducing affinity of enzyme active sites. It is known that cellwalls of green algae contain trace amounts of an unidentifiedinsoluble component believed to play a role in maintainingthe organization of cellulose fibers (21). Variation in suscep-tibilities of various genera of green algae to chemical andbacterial decomposition may be a function of the amount ofparaffinic structures associated with the cellulose in cell wallfibrils. As such, paraffinic structures could provide algal cellswith immunity to breakdown and catabolic metabolism byepiphytic bacteria. Thus, synthesis of macromolecular par-affinic structures would be an ecological advantage for greenalgae.

In summary, it is concluded from results of this study thatmembers of the Chlorophyta contain an insoluble structurecomposed of paraffinic carbon that is most likely associatedwith cell wall components. This paraffinic structure appearsto be resistant to bacterial and chemical decomposition andtherefore able to be incorporated into sediment. Paraffinicstructures of alga-derived hydrolyzed alghumin demonstratea chemical structure similar to that of humin in sediments ofaquatic origin, as shown by solid-state 13C NMR spectros-copy.

ACKNOWLEDGMENTS

This work was supported in part by the U.S. Geological SurveyG. K. Gilbert Fellowship 1981-1983 and Environmental ProtectionAgency cooperative agreement no. CR812246-01-0.We thank E. Spiker, U.S. Geological Survey, for isotope analysis,

helpful discussion, and review of the manuscript and R. Sjoblad forthe culture of D. tertiolecta used in this study. The excellenttechnical assistance of W. d'Angelo and Z. Brown, U.S. GeologicalSurvey, E. Karlander, University of Maryland, and N. Szeverenyi,Colorado State University, is gratefully acknowledged. The authorsalso acknowledge the late Irving A. Breger for his friendship and hisguidance and stimulating discussion in this research endeavor.

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