Cellulose degradation by a new isolate from sewage sludge, a member of the Bacteroidaceae family1 Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MICHIGAN on 11/21/14 For personal use only.

J . N. SADDLER A N D A . W. KHAN Dirisiotr cf! Biological Sciolces, Ntrfiot~crlResccrrch Cororc.il of 'Ctrrrcrcicr, Ott(i~i~tr, Otlr., Catrcrcitr K I A OR6 Accepted August 28. 1979 S A D D L E JR. . N., and A. W. K H A N .1979. Cellulose degradation by a new isolate from sewage sludge, a member of the Bacteroidaceae family. Can. J . Microbiol. 25: 1427-1432. A mesophilic anaerobe. a member of the Bacteroidaceae family (NRC2248), isolated from a cellulose-enrichment culture. digested untreated Whatman cellulose powder and HCI-treated cotton battings while producing hydrogen, carbon dioxide, cellobiose. glucose. and acetic acid as the sole volatile acid. This organism also utilized cellobiose as carbon and energy source but did not utilize glucose. I t grew well in synthetic medium containing ammonium salts as nitrogen source and having a pH value of 7.0-7.1 and an El, value of - 160mV or lower. The n~rtrient requi~.ementsof this or-ganismwere found to be similar to those of other anaerobes except fo~.NazS which inhibited cellulose degradation in concentrations above 0.75 mM. Best cellulose degradation occurred under an atmosphere of 80% N, - 20% CO?. Use of HZ or 80% H2- 20% CO, a s headspace gas inhibited growth. Although accumulation of acetic acid in about 15-16mM concentrations inhibited the further formation of H Z ,CO?, and acetic acid in the broth. it did not stop the degradation of cellulose. The results indicate that this organism has the ability togrow in media containing up to 2Og/L of cellulose and to produce industrially important and easily separable end products from cellulose. SAI)DLER. J . N.. et A. W. KHAN.1979. Cellulose degradation by a new isolate from sewage sludge, a member of the Bacteroidaceae family. Can. J . Microbiol. 25: 1427- 1432. Un mesophile anaerobe, membre de la famille des Bacteroidaceae (NRC 2248), en provenant d'une culture d'enrichissement sur cellulose, a degrade la poudre de cellulose Whatman non traitee et des touffes de coton traittes au HCI avec une production d'hydrogene, de dioxyde de carbone. de cellobiose, de glucose et d'acide acetique comme seul acide volatile. Cet organisme utilisait aussi le cellobiose comme source de carbone et d'energie mais pas le glucose. II croissait bien dans un milieu synthetique contenant des sels d'ammoniac comme source d'azote sous un pH de 7.0-7.1 et un E,, de - 160mV ou moins. Les exigences nutritives de cet organisme etaient semblables a celles d'autres anaerobes, exception faite du Na,S qui, en concentrations superieures 0.75 mM, inhibait la degradation de la cellulose. La meilleure degradation de la cellulose se produisait sous une atmosphere de N, a 80% et de CO? a 20%. L'utilisation de H, ou de Hz a 80% et de CO? a20% dans I'espace de t6te inhibait lacroissance. MGme si I'accumulation d'acide acetique a des concentrations de 15- 16mM inhibait la formation ulterieure de H , d e C 0 2 et d'acide acetique dans le molit, elle n'arrktait pas la degradation de la cellulose. Les resultats indiquent que cet organisme posstde la faculte de croitre dans des milieux contenant jusqu'h 20glL de cellulose et d'y produire des derives d'importance industrielle et facilement recuperables. [Traduit par le journal]

Introduction cellulose degradation by this bacterium and the During the study of actively celluloytic bacteria factors influencing its cellulolytic ability. present in a mixed culture enriched on synthetic Methods and Materials medium containing cellulose (Khan 1977), an anaerobic. Gram-negative, motile, rod-shaped The isolation and characterization of this organism (NRC bacterium was isolated. Most of the anaerobes so far isolated in pure culture (a) do not ferment cel- 2248) will be described in detail elsewhere. In brief, this isolate is similar to Bacferoicies .sirccitrogrties (Bryant and Doetsch lulose in large concentration, (b) have exceedingly most 1954; Hungate 1950) but differs in that it does not ferment slow growth in media containing cellulose, or (c) glucose o r produce succinic acid. This organism was maintained form a large diversity of products, and thus have in a chemically defined medium, previously described by Khan been considered unsuitable for industrial purposes et al. (1979). and containing (milligrams per litre in parentheses), (Hungate 1950). Since this anaerobe formed easily NaHCO, (2060); NH4CI (680); KZHP04(296); KH2P04(180); (NH4)?S04 (150); MgS0,.7H20 (90); CaCI2.2H,O (60); separable end products, tests were made to study FeS04. 7 H Z 0 (21); N(CH2COOH), (IS); MnS04. H,O (5); 'NRCC No. 17856.

CoCI?. 6H,O (1); ZnS0,. 7HZ0 (1); CuSO,. 5H,O (0.1); AIK(S04)Z 12H,O (0. I); H 3 B 0 3 (0.1); NaZMo04.2 H 2 0 (0.1);

0008-4 166179112 1427-06$01 .N/O @ 1979 National Research Council of CanadaIConseil national de recherches du Canada

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C A N . J . MICROBIOL. VOL. 2 5 . 1979

pyridoxine HCI (0. I); thiamine HCI (0.05): riboflavin (0.05); nicotinic acid (0.05); p-aminobenzoic acid (0.05); lipoic acid (0.05); biotin (0.02); folic acid (0.02). and vitamin B12 (0.005). This medium also contained cysteine-HCI (250mgIL). and N a z S . 9 H z 0 (750mgIL) ;IS reducing agents (Holdeman and Moore 1973). resazurin solution (I mg/L. 0.1% solution) a s redox potential (E,,) indicator, and cellulose ( I g / L ) . The medium was prepared following the procedure of Holdeman and Moore (1973). The pH was adjusted to 7.2-7.4. prior to being reduced (Hungate 1950). The reduced medium was dispensed in 10-mL volumes to 60-mL serum vials containing plwveighed amounts of cellulose and autoclaved at 15 psi (I psi = 6.894 757 Pa) for 15min. The substrates used were cellulose powder CF-11 (Whatman), acid-washed microcrystalline cellulose (Avicel). and D-(+) cellobiose (Eastman Kodak). All solutions and water used in these tests were equilibrated and bottled under 80% NZ- 20% COZ mixture. T h e headspace of the vials also contained the same gas mixture. These vials were inoculated and incubated at 37°C with shaking. This culture was maintained by weekly transfers and a 3-day-old culture was used as an inoculum in 2% (vlv) concentrations. The effects of iron (FeCI,). sulfate, and reducing agents present in the medium on the degradation of cellulose were studied by deleting the desired material from the medium and by adding it back in varying concentrations in the form of FeCI,, N a z S . 7 H z 0 , NazS04. and cysteine.HC1. pH studies were carried out using0.2 M citrate (pH 3-6). 0.2M phosphate (pH 6-8). and 0.2 M universal buffers (pH 4-7). The effects of various gases o n cellulose degradation were studied by using COZ,H z . N,. o r a mixture of these gases in the headspace of the vials. T h e volume and composition of gases produced from cellulose degradation were corrected for initial headsp;~cegas. In all cases. the total volume of medium in each vial was 10rnL. Results were averaged for at least three vials and were repeated on two different occasions. Growth was assessed by measuring the optical density at 620nm in a cuvette of a 10-mm light path. Gas volume was measured by gas manometer. Gas composition was assayed according to van Huyssteen (1967) and volatile acids according to Ackman ( 1972) using chromatography. Details of these methods have been reported earlier (Khan and Trottier 1978). For analysis. the total contents of each vial were centrifuged at 6000 x g for 20 min. T h e supernate obtained was used for the estimation of volatile acids and reducing sugars. Total reducing sugars were assayed by both the anthrone method of Herbert et al. (1971) and the dinitrosalicylic acid method of Miller (195911). Glucose was assayed using the glucostat method (Sigma) and measuring the oxidation of glucose to gluconic acid (Raabo and Terkildsen 1960). Two-dimensional paper chromatography on Whatman paper No. 1 using solvent systems butanol :ethanol : water (40 : 1 1 : 19, vlv) and ethyl acetate :acetic acid : water (3 : 3 : 1, vlv) was also employed for the detection of sugars produced from cellulose and cellobiose degradation. Since glucose and cellobiose were the only reducing sugars present when cellobiose was used as the substrate, cellobiose content could be determined from the difference between the total reducing sugars and glucose contents. The pellet was treated with formic acid (Weimer and Zeikus 1977) and NaOH (Miller 1959n) to remove protein and washed by centrifugation. The pellet was dissolved in 72% H,SO, and the cellulose content was determined by the anthrone reagent

(Herbert et al, 1971). The major advantages of this method are ( ( I ) application of the anthrone reaction directly on cellulose solution in 72% HSO,. which is otherwise difficult to solubilize, and ( b ) presence of glucose and its di- and poly-saccharides including cellulose give quantitatively the same color value per mole of glucose; also their absorption spectra show a single main peak (Herbert et al. 1971). T o improve the accuracy further, the method was standardized for each type of cellulose used as a substrate.

Results The isolate grew in a medium containing cysteine HCI at concentrations of 0.1 to 40.0mM, equivalent to E hvalues of -50 to -300 mV (Fig. 1). Maximum growth was noted at 1 mM cysteine. HCI concentration, and at an Eh value of about - 160mV. Above these levels, cysteine had no beneficial effects on the growth. This organism was originally isolated from a mixed culture maintained on medium devoid of reducing agents (Khan et al, 1979). For the maintenance of pure culture, reducing agents normally used for the cultivation of anaerobes were included in the medium because of the fastidiously anaerobic nature of this organism. Since the presence of cysteine.HC1 above 1 mM concentration had no beneficial effect on growth (Fig. I ) , and since the presence of sulfide has been shown to inhibit cellulose degradation by mixed culture from which this organism was isolated (Khan and Trottier 1978), different combinations 'and concentrations of cysteine, sulfides, and sulfate were used (Table 1). In these tests, the Eh value of the medium was kept below - 125 mV by using 0.75 mM or higher concentration of cysteine (Fig. 1). The concentrations of cysteine in the range of 0.75-3.00 mM appear to have no detrimental effect on cellulose degradation or acetate formation. However. 1.0 and 1.5mM concentrations of Na,S in the presence of cysteine inhibited both cellulose degradation and

FIG. 1. Relation. between various levels of reducing agent: oxidation-reduction potential (a),and growth (0).

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TABLE 1. Effect of varying concentrations of sulfur supplied in the form of cysteine, sulfide, and sulfate o n cellulose degradation Cellulose utilizedb Sulfur source," m M

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% of initial Cysteine

Sulfide

Sulfate

value

mM

Acetic acid formed, mM

OUscd as cysteine,HCI, Na2S.7H20, and as inorganic sulfates. bMedium containing 5 g/L aviccl cellulose. Incubation time 3 days

acetic acid formation by about 10%. Both cysteine and Na,S were used in these tests, because a combination of these two materials was more effective in reducing the medium than either of these two materials alone. Combinations of the two reducing agents in concentrations lower than 0.75 mM of each were equally effective for efficient growth and cellulose degradation. However, it proved difficult to reduce the medium at these lower concentrations. Lowering the sulfate concentration of medium containing 0.75mM of cysteine and 0.75 mM of Na,S from 1.7mM to 0.1 mM resulted in a 20% decrease of cellulose degradation, whereas an increase from 1.7 to 2.5 mM produced a 10% decrease in the cellulose degradation. Acetic acid formation was correspondingly lower at these concentrations. An iron content of 0.6 mM in the medium gave the best results in degrading cellulose and in producing Hz, acetic acid, and reducing sugars (Fig. 2). Iron concentrations as low as 0.1 mM, and as high as 1.5 mM, caused only about 20% inhibition as compared with the optimum concentrations of these minerals. No attempt was made to study the vitamin and trace mineral requirements, and these materials were used in concentration normally recommended for the cultivation of anaerobes (Holdeman and Moore 1973). Growth and cellulose degradation by the organism occurred within a temperature range of 20-40°C and a pH range of 6.0-7.5, with temperature optimum at 37°C and pH optimum at 7.0 (Fig. 3).

I R O N CONCENTRATION (rnM)

FIG.2. Effect of iron o n cellulose degradation

(a).acetic acid

(0). Hz (0). CO, (D),and reducing sugars (A) formation. Initial cellulose concentration 5 g/L.

Tests made in serum vials containing 80% N, - 20% C 0 2 as headspace gases gave the best results for cellulose degradation (Table 2). Under an atmosphere of N, alone, the lag period ofgrowth was somewhat extended as compared with atmosphere containing 80% N, - 20% CO,. It may indicate that the isolate requires CO, in the initial stages of growth. After the fulfillment of this requirement, the growth under N, was comparable with that occurring under 80% N,-20% CO, or

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of avicel cellulose, and at 20gIL cellulose concentration it could degrade cellulose at a rate of 2 g/L day-'. At higher than 5 g/L cellulose concentration. after about 3 days ofgrowth, the formation of acetic acid at 15-16 mM level inhibited the formation of H,. CO,, or any further amounts of acetic acid. Although growth was inhibited at these acetic acid levels, the breakdown of cellulose continued, perhaps as a result of extracellular cellulase released by the organism.

TEMPERATURE ( O C I

PH

FIG.3. Effect of temperature (@) and pH (0) on growth.

CO, alone. On the other hand, an atmosphere of 100% H2 or 80% H, - 20% CO, inhibited growth. The ability of this organism to ferment cellulosic materials with different degrees of polymerization at different concentrations are shown in Table 3. At a concentration of I g/L, the degradation of both cellulobiose and avicel cellulose to acetic acid, CO,, and H2 occurred at approximately the same rate. After 3 days ofgrowth, the yields of H,. CO,, and acetic acid from these two substrates on a mole per mole basis were 4.0, 2.7, and 0.7 respectively. The reducing sugars released from avicel cellulose contained about 75% glucose and 25% cellobiose. Degradation of cellulose powder proceeded at a slower rate than did the degradation of cellobiose or avicel cellulose. At 1 g/L concentration after 3 days of growth, the yields of H,, CO,, and acetic acid obtained from cellulose powder were respectively 3.0, 2.0, and 0.5 on a mole per mole basis of cellulose utilized. Reducing sugars released contained about 50%glucose with the remainder consisting of cellobiose and short-chain polymers of cellobiose. The isolate grew in medium containing up to 50gIL TABLE 2. Effect of carbon dioxide, hydrogen, and nitrogen on cellulolytic activity

Gas phase

Incubation time, days

Cellulose digested," mM

Owhatman cellulose. Initial concenlration, 5 g/L, bT,traces or none.

Acetic acid formed, mM

Discussion The organism isolated from an enrichment culture originally obtained from sewage sludge showed appreciable growth in the temperature range of 20-40°C and pH range of 6.0-7.5 and has a temperature optimum at 37°C and a pH optimum at 7.0. Like other fastidious anaerobes, it required an E,, value of about - 100 mV for growth. Since this organism produced H, from cellulose, in an actively growing culture low E,, value conditions would be maintained. This organism grew well in the chemically defined medium originally designed for mixed culture from which it was isolated. The mineral and vitamin requirements appear to be basically the same as those of other anaerobes (Bryant, et al. 1959; Caldwell and Bryant 1966), except that this organism did not require an organic nitrogen source such as yeast extract or peptone, as well as growth-promoting factors such as are present in sewage sludge or long-chain fatty acids. Perhaps this anaerobe is unique among cellulolytic bacteria in utilizing an inorganic nitrogen source for the synthesis of essential amino acids and proteins a s well as in degrading cellulose in a chemically defined medium. Whether or not the Dresence of an organic nitrogen source or vitamins in concentrations other than used in these tests will improve cellulose degradation requires a separate investigation. The degradation of cellulosic materials resulted in the production of Hz, COr, acetic acid, and reducing sugars, mainly glucose and cellobiose. Although the accumulation of acetic acid is approximately 15- 16 mM concentration inhibited growth a s well as further production of acetic acid, H,, o r CO,, it did not prevent the breakdown of cellulose to cellobiose and glucose. It appears that the isolate produces a cellulase system which degrades cellulose to cellobiose; the organism then utilizes cellobiose by fermenting it to H,, CO,, aceticacid, and glucose. Since. the digestion of cellulose continued even after growth and fermentation of cellobiose had stopped as a result of low p H , cellulolytic and fermentative processes appear t o take place inde-

SADDLER A N D K H A N

TABLE 3. Degradation of various cellulose substrates at various concentrations Substrate utilised

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Substrate -

Incubation time, days

Initial cellulose content, g/L

Gas formed, n1M

% of initial amount m M

H,

CO,

Acetic acid formed, mM

Reducing sugars formed, rnM

-

Cellobiose

3 3 3

N.T." N.T. N.T.

Avicel cellulose

3 3 6 3 6 3

0.8 2.6 3.1 3.8 7.7 N.T.

Whatman cellulose powder CF-11

3 3 6 3

G 'N.T..

not tested.

pendently. The fact that Hz and acetic acid, the two major end products of cellulose degradation, inhibited the growth of the isolate indicates the reason for the existence of this organism in mixed cultures where H, and acetate are readily utilized. The role played by this or-ganism in producing glucose, a sugar which itself does not utilize. is not clear. Perhaps the formation of glucose stimulates the growth of some other microorganisms present in natural habitats which play an important symbiotic function. The results indicate that the organism degraded cellulose most effectively in medium containing 5- IOg/L of cellulose. In medium containing 20gIL of avicel cellulose, degradation occul-red at a rate of 2g/Lday - I . This organism could also grow in medium containing up to 50g/L of avicel cellulose. However, the degradation of cellulose powder occurred at a slower rate than did the degradation of avicel cellulose, probably as a result of a greater degree of polymerization of this substrate. Although mesophilic anaerobes capable of attacking cellulose are widely found in nature, only a few have been isolated in pure culture (Bryant 1959; Hungate 1966). Halliwell and Bryant (1963), in examining the breakdown of treated and untreated cellulose using one strain of B. succinogenes, two albr(s, and two strains of strains of R~lt7litlococc~ls R~rnlit~ococcus flacijiiciens, found that while all five strains degraded cellulose powder, only B. succinogenes could break down untreated cellulose, at a rate of about 0.4g/Lday-l. One of the most actively cellulolytic thermophillic organisms isolated so far, ClustriCii~~t71 thertnoc~'II~o~1, has been shown to degrade cellulose at about 0.9 g/L day-I (Weimer and Zeikus 1977). The growth of

most known cellulolytic anaerobes is inhibited when about 1-3g of cellulose per litre is digested (Hungate 1950). During the work described here, no attempt was made to test the cellulolytic ability of this organism by minimizing the effects of end products such as H, and acetic acid which inhibit its growth. This approach should further improve cellulose digestion by this organism. Acknowledgement The authors thank Mrs. V. M. Laube for technical assistance. A C K ~ I AR. N .G. 1972. Porous polymer lead packings and formic acid vapor in GLC of volatile free fatty acids. J . Chron~atogr. Sci. 10: 560-565. B R Y A N TM. . P. 1959. Bacterial species of the rumen. Bacteriol. Rev. 23: 125- 153. BRYANT. M. P., and R. N . DOErsCH. 1954. A study of actively cellulolytic rod-shaped bacteria of the bovine rumen. J . Diary Sci. 37: 1176-1 183. B R Y A N TM. , P.. I. M. ROBINSON, and H . C H U . 1959. Observations on the nutrition of Brrc~eroidcs.slrccitiogetrc.s. A ruminaI cellulolytic bacterium. J . Dairy Sci. 42: 183 I- 1847. C A L I > W E LD. L ,R.. and M. P. BRYANT.1966. Medium without rilmen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 14: 794-801. H A L L I W E L G.. L , and M. P. BRYANT. 1963. The cellulolytic activity of pure strains of bacteria from the rurnen ofcattle. J . Gen. Microbiol. 32: 441-448. HERBERT, D.. P. J . PHIPPS. and R. E. STRANGE. 1971. Chemical analysis of microbial cells. Itr Methods in microbiology. Vol. 5B. Erlilcd hy J . R. Norris and R. W. Ribbons. Academic Press. New York. pp. 209-344. HOLDEMAN L.. V., and W. E. C. MOORE.(Erlilors.) 1973. Anaerobe laboratory manual. 2nd ed. Virginia Polytechnic Institute, State University. Blacksburg. H U N G A T ER., E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14: 1-49. 1966. The rumen and its microbes. Academic Press. New York.

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K H A N . A. W. 1977. Anaerobic degradation of cellulose by -195%. Use of dinitrosalicylic reagent for determinzltion of reducing sugars. Anal. Chem. 31: 426-428. mixed culture. Can. J . Microbiol. 23: 1700-1705. 1960. . On the enzymatic . Effect of sulphur- RAABO.E., and T . C. T E R K I L D S E N K H A N .A. W.. and T . M. T R O T T I E R1978. determination of blood glucose. Scand. J . Clin. Invest. 12: containing compounds on anaerobic degradation of cellulose to methane by mixed cultures obtained from sewage sludge. 402-406. V A N HUYSS'I-EEN, J . J . 1967. Gas chromatographic separation of Appl. Environ. Microbiol. 35: 1027-1034. digestergases usingporous polymers. WaterRes. 1:237-242. K H A N ,A. W.. T . M. TRO-I-TIER, G. B. PATEL,and S. M. MAR. J.. and J . G . Z E I K U S1977. . Fermentation of celT I N . 1979. Nutrient requirement for the degradation of cel- W C I M E RP. lulose and cellobiose by C'lostridiroi~tl~ertt~occllritr~ in the lulose to methane by a mixed pop~lationofanaerobes.J . Gen. tlzertnocrrrtoabsence and presence of Mctl~trt~o/>crcterirrt~~ Microbiol. 112: 365-372. M I L L E RG, . L. 1959tr. Protein determination for large numbers t r o p l ~ i c r r rAppl. ~. Environ. Microbiol. 33: 289-297. of samples. Anal. Chem. 31: 964-966.

Cellulose degradation by a new isolate from sewage sludge, a member of the Bacteroidaceae family.

Cellulose degradation by a new isolate from sewage sludge, a member of the Bacteroidaceae family1 Can. J. Microbiol. Downloaded from www.nrcresearchpr...
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