Indian Journal of Experimental Biology Vol. 39. June 200 I, pp.584-589

14C-[lignin]-lignocellulose biodegradation by bacteria isolated from polluted soil

Lokesh Kumart, YS Rathore* & HS Srivastava**

*Department of Environmental Sciences, College of Basic Sciences & Humanities, **G. B. Pant University o f Agricu lture and Technology, Pantnagar 263 145, India

tDepartme nt of Plant Sciences, Faculty o f Life Sciences, Rohilkhand University, Bareilly 243 122, India

Received 25 August 1999; revised 6 February 2001

Four bacterial species [Branhamella catarrhal is (gram -ve). Brochothrix species (gram -ve), Micrococcus /uteus (gram +ve) and Bacillus firm us (gram +ve)] , isolated from the soi l polluted with cane sugar factory efn uents. were fo und capable of growing on solid media supplemented with indulin AT (a polymeric industri al li gnin ) as sole C source. All the four species cou ld metabolize ci nnamic ac id (a non-hydroxy lated phenylpropanoid) as sole carbon source wi th significant suppression on addi tion of readi ly metaboli zable carbon source (glucose). However, Br. catarrhalis and Brochothrix sp. were capable of metabolizing ferulic ac id. but could not do so on addition of g lucose. Of the four species, Br. catarrhal is cou ld evolve significant amount of 14C02 from U- 14C (li gnin)- lignocellulose prepared from rice stalks (ca. I 0% of the added radioactivity in 3 weeks), in addition to solubi li za tion of another I 1.7% radioactivity in culture filtrate. The other three species could not significantly evolve 14C02, though a significant fraction of added 14C- Ii gnin (6. 1 to I 1.2%) could be solubi li zed into culture filtrate, suggesting lack of ring-cleavage or other C0 2 evolving mechanisms in these spec ies.

An estimated one hundred billion tonnes of terrestri al plant biomass is produced annually, of which lign in accounts for at least 20 billi on tonnes, making this biopolymer the biggest source of benzene nucleus in the biosphere1.2. However, this vast renewable resource remains largely unused due to close association between the plant cell wall polysaccharides and lignin , and highly recalcitrant nature of the latter3

.

Utilization of this potential resource for such diverse uses as production of ethanol, sugars and proteins fro m cell wa ll polysaccharides, novel aromatic compounds from lignin , upgrading of straw for cattle feed, and improvement in pulping and bleaching processes, requires identification of efficient ligninolytic microorganisms as well as detailed knowledge of biochemistry and enzymology of delignification and its b.iodegradation4

.

Earlier studies in this field have been impeded due to lack of specific assays for lignin biodegradation. However, development of methodology for preparation of specifically radio labelled plant lignins and synthetic lignins (DHPs) coupled with quantitative radioassays for li gnin mineralization5

, have resulted in great upsurge in research on lignin biodegradation . Most of the avai lable literature on lignin biodegradation is confined to a few genera of white- rot fungi like Phanerochaete chrysosporium and Trametes versico!ol -8. Bacteria, in particul ar, deserve to be

studied for lignino lytic potential, because of their immense environmental adaptability and biochemical versatility. Earlier studies have shown that some bacterial species could grow on lignin or lignin related compounds as sole carbon and energy source9

-10

.

Several strains of Nocardia and Pseudomonas 11,

Arthrobacter12, Xanthomonas13

, and mixed bacteri al culture from lignin-rich natural habitats 14 have been reported to evolve 14C02 from dehydropolymerizates (synthetic lignins) or natural lignins, specifically rad iolabelled with carbon-14 in methoxyl (-0 14CH3),

propyl side chain (2' or 3' labelled) or benzyl ring of the lignin molecule or dimeric, trimeric and tetrameric model compounds structurally-related to Iignin 15

-17

.

Ultrastructural evidence has supported these biochemical findings 18. However, the ligninolytic rates of reported bacteri al species have been found to be much lower compared to lignin-degrading basidiomycetous fungi such as Phanerochaete chrysosporium . For thi s reason there is need for continued search for more efficient ligni no lytic bacterial species which can be used sing ly or in consorti a to develop lignin-biodegradation technologies for specific and diverse uses. In the present paper we have reported isolat ion and characterization of ligninolytic potential of four bacteri al species isolated from soil polluted with sugar factory effluent.

KUMAR eta/.: 14C-[LIGNIN]-LIGNOCELLULOSE BIODEGRADATION 585

Materials and Methods Source of chemicals-(U) L- 14C-phenylalanine

(sp. act.: 450 milliCi/mmole) was obtained from Bhabha Atomic Research Center, Trombay, India. Indulin AT, chymotrypsin, cinnamic acid, ferulic acid and HPLC reference standard compounds were obtai ned from Sigma Chemical Co. , USA.

Isolation and identification of bacteria-Soil samples (upper 3") collected from the bank of effluent canal emanating from RBKS Sugar Factory, Bilaspur Dist. Rampur, India., were subjected to isolation of predominant bacterial strains by serial dilution, followed by spreadi ng on the basal mineral medium 19, supplemented with purified Kraft lignin (PKL, 0. L% w/v)20

. The isolated bacterial species were identified by morphological, physiological and biochemical tests21 in collaboration with the Institute of Microbial Technology, Chandigarh. The isolated bacterial species were maintained on multiple carbon nitrogen source agar22

Metabolism of lignin related aromatics­Metabolism of ferulic and t-cinnamic acid as the sole carbon source by the isolated bacteria was studied in presence and absence of glucose according to Falcon et a/. 23

. Optical density of the culture filtrate was recorded at 288 and 279 nm using UV-VIS spectrophotometer at 24 hr intervals up to 4 days, respectively.

Assayfor14 C( I ignin)l ignocelluloseeg radat ion- 14C­(Iignin)-lignocellulose (specific activity = 5.5x 103

dpm/mg) was prepared from 4 week old rice seedlings, according to Crawford and Crawford24 .

Radiorespirometric assay for lignin mineralization (14C02 evolution) and solubilization e4C- products in culture filtrate) was performed in duplicate using I 00 mL Erlenmeyer flasks containing 25 mL basal mineral medium25

. 14C-lignocellulose [chymotrypsin-treated;

I xI 05 dpm (20 mg)] was added as substrate in each flask. 14C0 2 released was trapped in scintillation vials each containing LO mL of Bray's scintillation fluid26

supplemented with LO% ethanolamine (to absorb 14C02), at 3 day intervals by passing C02 free sterile air. Radioactivity was counted with a liquid scintillation counter (about 90% counting efficiency) equipped with external quench correction.

Biodegradation products-HPLC, was performed 600 E high pressure liquid chromatograph using a 484 UV detector on a waters and 745 B data module. A bonda pak octadecylsitane (C 18) covalently-bonded to

a silica solid support of 5 J..lm pore size, reverse phase column (3.8 mm i.d. x 30 em) was used. The chromatograms were read at 280 nm at a chart speed of 0.5 em/min. The samples of culture filtrate fo r HPLC analysis were extracted and prepared according to Chen et al.,21

. A 5J..1L sampling loop was injected followed by implementation of gradient for solvent delivery using water (pH 3.2) containing acetonitrile initially at I mL min·1 flow rate (gradient : init ial : acetonitrile/water 10/90 flow rate I mL min-1; increase upto 5 min 25175, 2 mL min·1; after 5 min 25/75, 0.6 mL min-1; 12 min, 50/50, 0.6 mL min-1; 12-14 min, 50150, 0.6 mL min-1; 14-22 min, L0/90, 0.6 mL min.1). Identification of the compounds was performed by comparison of RT values with those of authentic samples and by co-chromatography .

Results and Discussion Isolation and characterization of bacteria -Four

predominant bacterial colonies based on morphological, physiological and biochemical tes ts were identified as Branhamella catarrhalis, Brochothrix sp., Micrococcus luteus and Bacillus Jirmus (Table 1).

Metabolism of phenylpropanoic acids-All the four species could utilize cinnamic acid (40-42%), when it was given as the sole carbon source (Table 2) . However, when glucose (55.5mM) was added in the culture medium, utilization of cinnamic acid was considerably suppressed (only 4.5 to 19.5 % utilization), depending on the species. In case of ferulic acid, only Br. catarrhalis and Brochothrix sp. could utilize it efficiently (78 and 52% respectively), in absence of glucose only. This seems to be due to the fact that glucose is a more readily metabolizable carbon and energy source and the cultures preferred it over the phenolic compounds. A similar preference for glucose and other readily metabolizable C sources has been reported in case of ligninolytic white-rot fungi 28

.

In fact, it has been found that the fungi do not produce lignin-degrading enzymes until the exogenously added glucose is substantially depleted from the culture medium29. For the other two species, ferulic acid proved to be a poor substrate, both in presence or absence of glucose. It has been reported that Nocardia sp. utilized 14C-ferulic acid more efficientl/0 than 3-0H-cinnamic acid as has been also observed for ferulic acid in the present study for Br. catarrhalis and Brochothrix sp.

586 INDIAN J EXP BIOL, JUNE 200 1

Table 1- Morphological. physiolog ica l and biochemical characteri za tion of the bacteria isolated from the so il s affected by sugar factory effluen t

Tests Coltmy Moi/Jlwlogv Configuration Margin Elevat ion Surface Pigment Grams Stain ing Shape Spores Motilit y Crmnh m Tc111pera111re 4"C I O"C 25"C 30"C 37"C 45°C SS"C Cro ll'lh at p/-1 5.7 6.8 8.0 Cro H'lh at NaCI 2.5% 5.0% 7Yk

Smooth undulate

rai sed glistening

-ve Cocci

±

+ + + +

+ + +

+ + +

10.0% + Acid production ji-0111 Carbohydrates

Smooth regul ar convex

glis tening

-ve Rods

+ + + +

+ + +

+ w

Glucose + Arabinose Lac to~c

Mannitol Sucro c Xylose Fructose Maltose Usc of ni1rogen Sou rces L-Phenylalanine Urea Hydrolysis En:y111e aclil'itv Nitrate reduction H2S Production Catalase Special Tests Growth on MacConkey Agar Citrate Utili za tion Starch Hydrolysi s Casien Hydrolysis Indole Test Methyl Red (M R) Voges proskour (VP) Gelatin Liquefaction Anaerobic Growth Oxidation/Fermentation Gas Prod uction

+ +

+ +

+ +

+ + + +

+

+

+ +IV

-I- -I-

Smooth regular convex

glisten ing

+ve Cocci

+ + + ±

+ + +

+

+

+ + +

+

-1-

Rough curled

umbonate gli sten ing

lemon yellow +ve

Rods in chains + +

+ + + + +

±

+ +

±

+ + +

+

IV

+

+ +

+

+

+ +

+ +

+I+

(+) Postil'e test (-) egative test , and (w) Weak test; 8 1 Brrmha111ella cmarrlwlis; 13J- M. luleus and 8 2 Brochothrix sp; and B4.B.jir111us

KUMAR et a/.: 14C-[LIGNIN]-LIG 10CELLULOSE BIODEG RADAT ION 587

Transformation of t.J C-( lignin )-lignocellulose-Defi­niti ve test of ligni nolytic capabilities of these species has been performed by studying the degradation of (U) 14C-(l ignin)- lignocellulose (Table 3). It can be seen that Br. catarrhalis was the most efficient of the four species. It released 9.9% of the added radioactive 14C­li gnocel lulose as 14C0 2 in 2 1 days and was still acti vely mineralizing lignin. In addition, 11 .7% .radioactivity of 14C-lignocellulose was recovered as soluble products in the culture filtrate in thi s species, Thus, a total of 21.6% of the added 14C-(li gnin)­li gnocellulose was subjected to biodegradation by thi s species during the 3 weeks period. This was quite comparable to 14C0 2 evolution from 14C-plant lignins or 14C-DHPs reported for other bacteri al species (Table. 4.). The other three species, howev.er, evolved relatively lower 14C0 2 (only 0.6 - 1. 1 %), but could solubili ze 14C-(lignin)-li gnocellulose appreciably (6.1 to 11 .2%). However, it may be noted that Broclwthrix sp. and B. j/m1us could solubilize as much as 11.2 and 8.3% of the added 14C-lignocellulose. Therefore, it would be interest ing to study the effect of combination of Br. catarrhalis and B. jlnnus or Brochohix sp. on lignin biodegradat ion in mixed culture experiments.

Thus, Br. catarrhalis , which was most effici ent in utilization of phenylpropanoid acids also showed highest mineralization of 14C-(lignin)-lignocellulose. A similar correspondence between phenylpropanoid metaboli sm and ability to mineralized 14C-DHP or 14C­(Lignin)- li gnocellulose has been reported for a number

f h I. . d d" . . !8?1 o ot er 1gnm egra mg microorgani sms ·-· . It may be pointed out that the time course of 14C02

evolution from 14C-(lignin)-lignocellulose substrate by Br. catarrhalis showed an initial lag phase of about 3 days, after which the rate increased substantially (results not given). A similar kinetics of 1 4CO~ evolution has been reported for other bacterial species5

and P. chrysosporium6·28

·29

, from radi olabelled plant lignin or 14C-DHPs. Further Br. catarrlwlis was found to be most effici ent li gnin-degrader belonging to gram -ve species, as reported for most of the other efficient li gninolytic bacteria so far8

.

Identification of lignill biodegradation products­Two products identified at RT, 2.60 to 2.69 (ga ll ic acid) and 7.33 to 7.85 (syri ngic acid) were present in the acid ified ether-soluble fracti on from the cu lture filtrate of all the four bacteria. Bacillus j/rn1us showed a maximum of 17 products in culture filtrate, among

Table 2 - Ulili za lion of c innamic acid and feruli c acid inlhe growth med ium by bacteria in presence (G+) and absence (G") of g lucose (55.5 mM )

[Values are mean of 2 repli cates]

Organism % utili zation (in hr)

24 48 72 96 24 48 72 96

Cinnamic acid Ferulic acid

Bralllwmella catarrhal is G+ 5.6 7.9 10.0 8.0 3 1.0 32.0 54.0 66.0 G. 16.5 27.5 40.0 42.0 21.0 57.5 72.0 78 .0

Brochothrix sp. G+ 11.0 14.0 11 .0 15.0 8.0 14.0 21.0 23.0 G. 16.0 26.0 42.0 42.0 13.0 44.5 47.0 52.0

Micrococcus !/Ileus G+ 15 .0 16.0 16.0 19.5 9.0 15.5 26.0 27.0 G- 7.5 8.0 42.0 42.0 14.5 I4.5 18.0 29.0

BacillusfimliiS G+ 4.5 4.7 4.7 8.5 16.5 14.5 20.0 27.0 G- 19.0 24.0 39.0 40.0 12.0 13.0 13.0 22.5

Table 3- 14C li gnocellulose degradation* by the bacteria isolated from the soil s affected by sugar factory effluent.

[Values are mean ± SD of2 rep licates!

Bacteri a Per cent of total 14C- added per culture

Bra11hamel/a catarrhalis

Brochothrix sp.

Micrococcus //Ileus

14C02 evolution

9.90 ± 1.21

0.60 ±0.05

1.00 ± 0.34

14C- in cu lture filtrate

11.70±1.60

11.20 ±5.72

6 .1 0 ±1.50

Baci/lusfirmus 1.10 ± 0.23 8.30 ± 1.84

After 2 1 days of incubation

*14C02 evoluti on in control sa mples were negli gible (< I%).

Total lignin degradat ion

2 1.60

11.80

7.10

9.40

588 INDIAN J EXP BIOL, JUNE 2001

Table 4-Lignin biodegradation rates by various bacterial species isolated from diverse natural habitats

Bacteria l Species Source/type of li gn in Incubation 14C02 evolution (as % of total added) Reference period (d) Position of 14C-in li onin

Propyl side Methoxyl Ring U-['4C}

Nocardia sp. DSM I 069 Maize Sta lks [14C]-lignin 15 Nocardia sp. DSM I 069 [

14Cj- DHP* 15 Noca rdia au101rophica [

14CJ- DHP 15 Nomrdia carol/ina [

14Cj - DHP 15 Nomrdia g /oberu/a [

14C]- DHP 15 Nocordia opaca (

14C]- DHP 15 Pseudolllonas pill ida [

14Cj- DHP 10 P. res1os1eroni [

14C]- DHP 10 Pseudolllonas I 06 U - [

14C] wood meal 15 U- [

14C] poplar wood 15

AnhrobaCier sp. U - J14C] cord grass 10 U- [

14C] slash pine 10 Salt marsh sediment U - [

14C] cord grass 23 consort ium Xamho111onas sp. 99 [

14Cj- DHP : m.wt. 850 14 m. wt. 1100 14 m. wt. 2400 14

Br. cawrrhalis U- [14C] paddy stalk 2 1

Bacillus fi n n us U - [14C] paddy stalk 21

Broch01hrix U- [14Cj paddy stalk 21

Micrococcus luleus U- J14Cj paddy stalk 21

*DHP - Dehydrogenated polymerisate (Synthetic lignin )

which seven products were identified as gallic ac id (RT: 2.60- 2.67), p-catechuic acid (RT: 3.60), sy ringic ac id (RT: 7.33-7.85), guaiacol (RT: 9. 17-9.73), p­coumaric acid (RT: I 0.50), veratraldehyde (RT: 13.83) and cinnamic acid (RT: 17.03). However, in Bmnlwmella catarrhalis, on ly seven peaks were recorded out of which four were identified as gall ic acid (RT, 2.60-2.69), p-hydroxy benzoic acid (RT: 4.64), syringic acid (RT: 7.33-7.85) and ferulic acid (RT: 10.97- 11.1 4). In case of Brochothrix sp. five products were found, out of wh ich three were identified as gallic acid, syringic acid and fe rulic acid. Seven products were recorded in Micrococcus luteus of wh ich four could be identified as gallic ac id , syringic ac id, guaiacol (RT: 9.17-9.73) and feru lic acid (RT: 10.97-11. 14).

Thus, Br. catarrhalis had in the culture fi ltrate all the three main constituents of grass lignin i.e. syringy l type (gallic acid and syri ngic ac id); guaiacyl type (guaiacol) , and p-hydroxyphenyl type (p- hydroxy­benzoic acid). This suggests that th is species possesses the capacity to break all the major types of bonds in the native lignin. It is also possible that rather than, or

chain

16 13 6 Trozanowski el a/ 30

7 II 4 Ibid 4.8--{).8 12. 1- 14. 1 5.3-7.6 Haider e1 a/ !

5

2. 1 6.1 3.1 Ibid 1.7 4.5 1.0 Ibid 17 4.5 1.0 Ibid

0.2 0.8 0.2 Ibid 1.1 2.1 1.1 Ibid

<1.0 Odier el a/ 11

3.5 Ibid

2.9 Kerr el a/ 12

< 1.0 Ibid 10 Benner e1 alu

Kern and Kirk 13

28.4 23.7 1.3

9.9 Present Study 1.1

< 1.0 1.0

in addition to having the abil ity to break all the major types of bonds in the li gn in , Br. catarrhalis also has the abili ty to cleave esterified compounds from peripheral units of the lignin . In this connection , the find ings of Kern and Kirk 13 are re levant, who have reported that the molecular weight of sy nthetic lignins influences their biodegradation by Xantlwmonas species-minerali zat ion being greatest for lignin samples having the lowest molecular weights, and the low molecu lar weight portions being preferentially degraded. Therefore, It has been suggested that the bacterial cells do not secrete lignin-depo lymerizing enzy mes extracellularly as do white-rot fungi, and the li gni n degradation in the former occurs intrace llularly after the low molecular weight lignins enter through the bacterial cell wall. A similar conclusion has also been drawn by Jokela et a/. 31

. Further studies usi ng Xanthomonas species and Br. catarrhalis which have been shown to have relatively hi gher lign in mineralization rates (Table 4) are needed to characteri ze the localization and pathways of lignin degrading enzymes.

KUMAR et a/.: 14C-[LIG IN] -LIGNOCELLULOSE BIODEGRADATION 589

Finally , it may also be mentioned that to the best of our knowledge th is is the first report of significant lignin minera li zation by Branhamel/a catarrhalis.

Acknowledgement One of the author (VS R) is grateful to Department

of Atomic Energy , Govt. of India, for financia l support received for this work.

References I Bassham J A, Cellu lose as a chemica l and energy resource.

Biotech Bioeng Symp. 5 ( 1975) 9. 2 Crawford D L & Crawford R L, Microbial degradati on of

lignocellulose : The lignin component, App/ £ 11 viron Microbial, 3 1 ( 1976) 714.

3 Boominathan K & Reddy C A, Fu ngal degradat ion of li gnin : Biotechnologica l applications, in Handbook of applied mycology , volA. edited by D K Arora, R P. Elander and K G Muke1ji (Marcel Dekker Inc. ew York) 1992.763.

4 Eriks on K-E L. Blanchette R A & Ander P. Biodegradati on of li gnin, in Microbial and enzymatic degradation of 1\'0od and \\'Ood components. (Springer- Verlag, Berlin) 1990, 225.

5 Haider K & Trojanow ki J A, comparison of the 14C- labell ed DHP and corn stalk li gnin by micro and macro fun gi and bacteria, in Lignin biodegradation: microbiology, chemistry and potential applications. vol.l, edited by T K Kirk, T Hi guchi and H Chang (CRC Press, Florida. USA) 1980, Il l.

6 Buswell J A, Fungal degrada tion of lignin , in Handbook r~f applied mycologv.vo l.l , edi ted by D K Arora, B Rai. K G Muke1ji and G R Knudsen (Marcel Dekker Inc, New York) 199 1. 425.

7 Cullen D & Kersten P J. Enzymology and molecular biology of lignin biodegradati on. in The Mycota : Biochemisn:v a/1(/ molecular biology, vol. 3, edited by K Esser and P A Lanke (Springer-Verlag. Berlin) 1996.295.

8 Kuhad R C, Singh A & Eriksson K-E L, Mi croorgani sms and enzy mes in volved in the degradation of plant fibre cell wa ll. in Biotechnology in the pulp and paper industry, edi ted by K­E L Eriksson (Springer- Verl ag, Berlin, Heidelberg) 1997, 45.

9 Gonza lez B, Meri no A, Almeida M & Vi cuna R. Comparati ve growth of natural bacterial iso lates on various lignin re lated compounds. Appl E111•iron Microbial. 52 ( 1986) 1428.

10 Kilpi S, Himberg K, Y1jala K & Backstrom V. The degradation of biphenyl and cholorobiphcnyls by mixed bacteri:~ l cult ures. FEMS- Microbial £col, 53 ( 1988) 19.

II Odier E, Janin G & Montics B, Poplar lignin decomposit ion by Gram negati ve aerobic bacteria. App/ E111•iron Microbial. 41 (1981) 337.

12 Kerr T J. Kerr R D & Benner R. Isolation of a bacterium cap:~ble of degrading pean ut hull lignin. Appl E111•iron Microbial. 46 ( 1983) 120 I.

13 Kern 1-1 W & Kirk T K. Influence of molecul ar size and li gninase treatment on li gnin degradati on by Xanthomonas sp. strain 99. Appl Environ Microbial, 53 ( 1987) 2242.

14 Benner R. Newe ll S Y. Maccrubbin . A E & Hodson R E, Anaerobic biodegradat ion of lignin and polysaccharide components of lignocell ulose and synthetic lignins by sediment micro fl ora. Appl Environ Microbial. 48 ( 1984) 36.

15 Jokela J J, Pellinen M, Salkinoja-Salonen M & Brunow G, Biodegradati on of two tetrameric lignin model compounds by a mixed bacterial culture, Appl Microbial Biotechnol. 23 ( 1985) 38.

16 Vicuna R. Gonzalez V. Mozuch M D & Kirk T K. Metaboli sm of lignin model compounds of the ary l glycerol­~-ary l ether type by Pseudomonas acidovorans D.1· Appl Environ Microbial, 53 (1987) 2605.

17 Kato K, Kozak i S & Sakuranaga M. Degradation of lignin compounds by bacteria from termite gut , Biotecluwl Lei/. 5 (1998) 459.

18 Rhoads T L, Mikell AT Jr. & Eley A 1-1 , In vest iga tion on the ligni n-degrading ac ti vity of Serratia marcescens: Biochemical screen ing and ultra structural evidence. Can 1 Microbial, 4 1 ( 1995) 592.

19 Odier E & Montics B, Biodegradation of wheat ligni n by Xanthomonas, A111wles de / ' Institute Pasteur/Microbio/ogie. 129A ( 1978) 36 1.

20 Kadam K L & Drew S W. Study of lignin transformation by Aspergillus fwnigatus and wh ite-rot fun gi usi ng 14C-labclled and unlabelled Kraft li gnins, Biotecluw l Bioeng. 28 ( 1986) 394.

2 1 Sneath P 1-1 A, Endospore fo rm ing Gram positi ve rods and cocci. In Bergey 's Manual of Systematic Bacteriology, vol.2. ed ited by P 1-1 A Sneath , N S Mair, M E Sharpe & J G Holt (Williams and Wi ll iams, Baltimore) 1986. 11 0-L

22 Hunter-Cevera J C. Fonda M E & Belt A. Isolation of cultures. in Manual of Industrial Microbiology and Bioteclmo/og_\'. edited by A L Demain & N A Solomon (American Society for Microbiology. Washington DC) 1986.3.

23 Falcon M A, Rodri guez A. Carnicero A. Rega lado V. Percstelo F, Milstein 0 & Fuente G Dela. Iso lation of microorgani sms with lignin transformation potenti al from soil s of Tenerif Island. Soil Bioi!Jiochem , 27 ( 1995) 12 1.

24 Crawford R L & Crawford D L, Radioisotopic methods for the study of lignin biodegrada ti on, Dev Indus Microbio/, 19 ( 1978) 35.

25 Haider K, Trojanowski J & Sundman V. Screening of li gnin degrading bacteria by means of 14C-labelled lignin , Arch Microbiol, 119 (1978) I 03.

26 Bray G A, A simple effi cient liquid sc intill ator for counting aqueous solution in a liquid sc intill ation counter. Anal Biochem. I ( 1960) 279.

27 Chen C L, Chang 1-1 M & Kirk T K. Aromatic ac ids produced during degradation of li gnin 111 spruce wood by Phanerochaete cluysosporium, Hol~fo rschung, 36 ( 1982) 3.

28 Kirk T K, Schultz E, Connors W J, Lorenz L W & Zeikus J G. Influence of cu lture parameters on lignin metabo li sm of Phanerochaete clu)•sosporiu111 , Arch Microbial. 117 ( 1978) 277.

29 Fa ison B D & Kirk T K, Fac tors in vo lved in the regul ation of ligninase ac tivi ty in Phaneroc/111ete cluysosporium. Appl Environ Microbic.{, 49 ( 1985) 299.

30 Trojanowski J. Ha ider K & Sundman V. Decompositi on of 14C- labelled li gnin and phenols by a Nocardia sp .. Arc/; Microbial. I 14 (1977) 149.

3 1 Jokela J J, Pell inen M, Salkinoja-Salonen M & Brunow G. Init ial steps in the pathway for bacterial degrada ti on of two tetrameri c lignin model compound , Appl Environ Microbial. 53 ( 1987) 2642.