APPLiED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1978, P. 631-6380099-2240/78/0036-0631$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 36, No. 5

Printed in U.S.A.

Degradation of Tyrosine in Anaerobically Stored PiggeryWastes and in Pig Feces

SIERK F. SPOELSTRALaboratory ofMicrobiology, Agricultural University, 6700 EJ Wageningen, The Netherlands

Received for publication 24 August 1978

Radioactively labeled compounds that might be intermediates in the anaerobicdegradation of tyrosine were added to pig feces and to stored piggery wastes.Changes in the compounds were followed by using thin-layer and gas chromatog-raphy. In feces, p-cresol and 3-phenylpropionic acid were the end products oftyrosine metabolism; in anaerobically stored mixed wastes, phenol, p-cresol, andminor quantities of phenylpropionic acid were formed. Schemes were proposedfor the degradation of tyrosine in pig feces and in mixed wastes.

Research on the chemical composition of themalodor emitted by piggeries revealed that p-cresol, phenol, and 4-ethylphenol are importantconstituents of the bad smell (32). Because theorigin of these simple phenols was poorly under-stood, a research program was started on theformation of simple phenols in piggery wastes.

In a previous investigation (37) high levels ofp-cresol and moderate amounts of phenol and 4-ethylphenol were detected in anaerobicallystored piggery wastes; the origins of these com-pounds were studied. Some of the simple phe-nols in these wastes were present at the timethat excreta were voided. Upon anaerobic stor-age of the mixture of feces and urine, whichgenerally occurs in pits under slatted floors, pro-tein degradation proceeds and concentrations ofphenol and p-cresol increase (37). The simplephenols present in freshly voided feces and urineare believed to originate from microbial degra-dation of tyrosine in the intestinal tract (13).Formation of phenol from tyrosine has been

reported for many bacteria possessing the en-zyme tyrosine phenol-lyase (EC 4.1.99.2) (9, 11,15, 16, 26, 31). An alternate pathway leading tophenol, with decarboxylation of 4-hydroxyben-zoic acid as the final step, has also been proposed(6, 13).p-Cresol is produced from tyrosine via de-

carboxylation of (4-hydroxy)phenylacetic acid(HPAA) (22, 34, 39). Organisms reported to pro-duce p-cresol belong mainly to the clostridia (15,31), but non-sporeforming organisms have alsobeen mentioned (39).

It has been suggested that 4-ethylphenol,present in the urine of mammals, would also bederived from tyrosine (28). But Bakke (4, 5)concluded from experiments with rats that p-coumaric acid (PCA) in plant material offoddersis the precursor of 4-ethylphenol. The conver-

sion of PCA to 4-ethylphenol is performed bythe intestinal microflora of rats (2, 34). No or-ganisms have been described which produce 4-ethylphenol from tyrosine.The microbial degradation of tyrosine as part

of an anaerobic ecosystem has been studied inhuman feces (12), rat cecal contents (2, 3, 5), andsheep rumen (36). For these different ecosystemsthe available information shows a complex pat-tern of possible degradation routes. End prod-ucts of tyrosine degradation in the intestine seemto be simple phenols, whereas in the rumenaromatic acids are accumulating. In the workreported here tyrosine degradation was studiedin anaerobically stored piggery wastes and infreshly voided pig feces.

MATERIALS AND METHODSSamples. Feces, separated from urine, were col-

lected overnight from castrated male pigs (Dutch Lan-drace or Yorkshire) held in metabolism cages.Farm slurry was taken as a grab sample from farms

with storage of mixed wastes under slatted floors.Extraction procedure. For the estimation of phe-

nols and aromatic acids in slurry or feces, samples(usually 3 g) were adjusted to pH 8.5 and extractedthree times with double volumes of ether. The ethereallayers were combined, dried over anhydrous Na2SO4,and evaporated under reduced pressure to a volumeof about 1 ml (neutral extract). The pH of the ex-tracted sample was adjusted to a value of 2.0, and thesample was again extracted twice with double volumesof both ether and ethylacetate. The combined organiclayers were dried over Na2SO4, evaporated under re-duced pressure to dryness, and dissolved in about 1 mlof acetone (acid extract).Gas chromatography. The neutral extracts were

subjected to gas chromatographical analysis for thequalitative determination of phenol, cresol, ethyl-phenol, and tyrosol. The operational data have beendescribed (37).

Aromatic acids were analyzed as their trimethylsilyl631

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APPL. ENVIRON. MICROBIOL.

derivatives or as methyl esters. The dry acid extractwas supplied with 0.2 ml of a mixture of N,O-bis-(trimethylsilyl)acetamide (Pierce Chemical Co.) andchloroform (1:2, vol/vol). The reaction mixture was

allowed to stand for 20 min at room temperature andwas subjected to gas chromatographical analysis.Methyl esters were prepared with boron trichlo-ride-methanol reagent (Sigma). The gas chromato-graph used was a dual-column Varian 2440 modelequipped with flame ionization detectors. For the anal-ysis of trimethylsilyl and methyl derivatives of aro-

matic acids, a 3-m, stainless-steel column (outer di-ameter, 0.125 inch [ca. 0.32 cm]) packed with 10% OV-17 on Chromosorb W/AW was used. The column oven

temperature was 200°C; temperature settings for theinjection port and detector block were set 30°C higherthan the oven temperature. Nitrogen (15 ml/min) was

used as carrier gas. Hydrogen and air flows were

adjusted at 30 and 300 ml/min, respectively.Thin-layer chromatography. Neutral and acid

extracts were applied to 0.25-mm layers of cellulose(MN 300, Macherey Nagel) or Silica Gel G 1500 Ls254 with a UV indicator (Schleicher & Schuell). Fortwo-dimensional development, solvent 1 (1.5% formicacid) and solvent 2 (upper layer of benzene-aceticacid-water, 10:7:3) were used in that order. Silica gellayers were developed usually in one direction onlywith solvent 2. Cellulose chromatograms were sprayedwith either diazotized sulfanilamide or with Fast BlueB salt followed by a 10% Na2CO3 solution (38). Colorsof the various compounds produced by the sprays were

reported by Scheline (33).Media. Clostridia were grown in closed 1-liter se-

rum bottles with either medium A (peptone, 5 g/liter;yeast extract, 2 g/liter; tyrosine, 0.2 g/liter) or one-quarter-strength reinforced clostridial medium (Ox-oid) enriched with 0.2 g of tyrosine per liter.

Radiochemical experiments. Radioactive com-

pounds that were possible intermediates in the deg-radation of tyrosine were added to tubes of 14-mlcapacity containing 3 g of either slurry or feces dilutedwith 2 parts of water. The tubes were supplied withabout 0.30,mol (approximately 0.05.,uCi) of the test.substances. The added tyrosine and tyramine con-

tained 0.1 and 0.25 ,uCi, respectively. The tubes were

flushed with oxygen-free nitrogen, closed with Suba-seal stoppers and incubated at 20°C (farm slurry) or

37°C (feces). At the end of the incubation period, thesamples were extracted as outlined above. About 10IlI of the extract was supplied to thin layers of silicagel, which were developed with solvent 2. Distributionof radioactivity was determined by scraping the spotsinto scintillation vials. Nonradioactive reference com-

pounds were run together with the samples.Because phenol and p-cresol could not be separated

by thin-layer chromatography, the ratios of phenoland p-cresol formed from added radioactive test sub-stances were estimated by gas chromatography. Forthis reason 5 to 10 Il of the neutral extract was injectedin a gas chromatograph equipped with an effluentsplitter, thus offering the possibility of collecting 90%of a compound after separation on the OV-17 column.The compounds were collected in a glass capillarywhich could be attached to the outlet of the streamsplitter. The capillary was cooled with a mixture of

solid carbon dioxide and acetone. The collected frac-tions were washed with ether into scintillation vials.

Total activity in the neutral and acid extracts wasdetermined by using an internal standard (["C]tolu-ene) as correction for quenching. The liquid scintilla-tion counter used was the Nuclear-Chicago Mark Imodel.Chemicals. Tyrosol was prepared microbiologi-

cally by incubating Saccharomyces cerevisiae withtyrosine and sucrose, according to Ehrlich (14). Lab-oratory strain S3 was used.

4-Hydroxystyrene was isolated from the culture me-dium of a Clostridium sp., strain A, which had beenincubated with PCA at pH 5.7. The strain had beenisolated from pig feces and was identified as C. ghoni.The product was isolated by extraction with ether ofthe culture medium brought to pH 8.5, and, afterreduction of the ethereal layer, it was purified by gaschromatography. The nature of the product was con-firmed by mass spectrometry. 4-Hydroxybenzylalco-hol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid,HPAA, 3-(4-hydroxyphenyl)propionic acid (HPPA),PCA, tyramine, (4-hydroxyphenyl)pyruvic acid(HPPyrA), 3-phenylpropionic acid (PPA), and phen-ylacetic acid (PAA) were purchased from Fluka. DL-(4-Hydroxyphenyl)lactic acid (HPLA) was obtainedfrom Sigma.Radiochemicals. L-[ U-'4C]tyrosine and side-

chain-labeled [2-14C]tyramine were purchased fromthe Radiochemical Centre, Amersham, England. [U-14C]tyrosol, [UU-14C]HPAA, [U-_4C]HPLA, and [U-'4C]HPPA were prepared microbiologically as de-scribed below.

Cells of an overnight culture of Clostridium strainA, grown in 1 liter of medium A at 37°C, were har-vested and aseptically transferred to a 25-ml tube. Thecells were incubated under a nitrogen atmosphere at37°C for 1 week with 0.5 mmol of L-[U-_4C]tyrosine(20 MCi), 0.25 mmol of a-ketoglutaric acid, and 1.0 molof glycerine in 10 ml of 0.1 M sodium phosphate buffer(pH 7.0). The only products formed from tyrosineunder these conditions were tyrosol, HPAA, andHPLA. The results of these experiments will be dealtwith in a separate paper.

[U-'4C]HPPA was isolated from the spent culturemedium of C. sporogenes NCIB 10696. This organismwas shown to accumulate HPPA by Elsden et al. (15).The organism was grown in 50 ml of one-quarter-strength reinforced clostridial medium supplementedwith 0.5 mmol of L-[U-_4C]tyrosine (5 ytCi) at 37°C.

Tyrosol was extracted from the reaction mixture atpH 8.5 with ether. The aromatic acids were extractedat pH 2 with ether and ethylacetate. The residues ofthe dried organic layers were dissolved in a smallamount of acetone and applied to thin layers of cellu-lose, which were developed with solvent 2. The areaswith the desired products were scraped from theplates, and the scrapings were re-extracted with ace-tone. The nature of the products was confirmed by gaschromatography of trimethylsilyl derivatives and bytwo-dimensional thin-layer chromatography. UV lightwas used to detect spots on the silica gel layers with afluorescence indicator. The thin-layer chromatogramsof HPPA when observed under UV light showed im-purities which, however, did not carry label.

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ANAEROBIC DEGRADATION OF TYROSINE 633

The labeled substances were dissolved in water, andtheir concentrations were determined gas chromato-graphically in a subsample as trimethylsilyl deriva-tives.

RESULTS

Preliminary experiments (Table 1). Ex-periments were set up to get an iinpression ofthe pathways involved in tyrosine degradationleading to simple phenols. Possibly occurringintermediates were added to the wastes, whichwere analyzed semiquantitatively for simplephenols after overnight incubation. No forma-tion of phenol or p-cresol was detected with thequalitative method of analysis used when nosubstrate had been added to farm slurry or feces.To approach the temperatures of the ecosys-

tems, farm slurry was incubated at 250C andfeces were incubated at 37°C. Previous experi-ments (37) had shown that no substantially dif-ferent results were obtained when farm slurrywas incubated at 20 or 370C. Phenol was formedin farn slurry from tyrosine and in minor quan-tities from HPPyrA, whereas no phenol wasformed when these substances were added tofeces. In farm slurry as well as in feces, 4-hy-droxybenzoic acid was decarboxylated to phenol.Small quantities of phenol were also formedfrom 4-hydroxybenzyl alcohol and from 4-hy-droxybenzaldehyde. p-Cresol was produced froma greater number of substances than phenol; thetypes of compounds which were converted to p-cresol were similar with farm slurry and feces.

Moderate to large amounts ofp-cresol were pro-

duced from tyrosine, HPPyrA, HPAA, 4-hydrox-ybenzyl alcohol, and 4-hydroxybenzaldehyde.Minor quantities were found when HPLA was

added. Tyramine and tyrosol gave no detectablequantities ofp-cresol after overnight incubation,but large amounts were found when analyzedafter 1 week or more.

4-Ethylphenol was detected when PCA was

added to feces, but not when it was added tofarm slurry. 4-Hydroxystyrene was totally re-

duced to 4-ethylphenol.The influence of the pH of farm slurry and

feces on the formation of simple phenols was

studied by adding the above-mentioned precur-sors to the wastes mixed with sodium phosphatebuffer of the desired pH (final concentration, 0.1M) and incubating the mixture overnight. ThepH was controlled and adjusted, if necessary,

after the addition of the solution with the testsubstances. After fermentation, the pH never

deviated more than 0.1 U from the initial value.In farm slurry phenol was produced from addedtyrosine in the pH range 7.0 to 8.0, whereas thedecarboxylation of 4-hydroxybenzoic acid oc-

curred over the whole range studied (pH 6 to8.5), with slightly higher activities at the lowerpH values (Fig. 1). The optimum pH for theconversion of tyrosine and HPAA to p-cresolwas about pH 7.0 in farm slurry. In feces no

optimum was found, and p-cresol was producedover the whole pH range studied (Fig. 2). Theformation of 4-ethylphenol from PCA in feces

TABLE 1. Production ofphenol, p-cresol, and 4-ethylphenol from tyrosine and from possibly occurringinternediates of the anaerobic degradation of tyrosine upon incubation of these compounds with farm

slurries and feces'Producth

Substrate Phenol p-Cresol 4-EthylphenolFarm Feces Farm Feces Farm Fecesslurry slurry slurry

Tyrosine ++ - ++ +++4-Hydroxystyrene - - - - ++ +++4-Hydroxybenzaldehyde + + ++ ++4-Hydroxybenzyl alcohol + + +++ ++Tyrosol - - [+1 [+14-Hydroxybenzoic acid +++ +++Tyramine - - [+] [+1HPAA - - ++ +++PCA - - - -HPPA - - - - - -HPLA - - + + - _HPPyrA + - ++ +++None -

'Test substances were added in concentrations of about 1 mg/ml. The pH value was buffered with 0.05 Mphosphate buffer at pH 7.0. Stoppered tubes were incubated under a nitrogen atmosphere at 25°C (farm slurry)or 37°C (feces) and analyzed qualitatively after 20 h.

h Formed in small (+), moderate (++), and large quantities (+++). Symbol in brackets denotes that no p-cresol was formed after the normal incubation time, but it was formed after long-term incubation.

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(n

z

lr

cr

(am

50

40

30

20

10

0 06 7 8 pH

FIG. 1. Effect of pH value on the formation ofphenol from tyrosine (0) and 4-hydroxybenzoic acid(U) added to farm slurry. (0) No addition.

50 r(A- 40

zD

30

lx20ar 10.4

0

6 7 8 pH

FIG. 2. Effect ofpH value on the formation of p-cresol from tyrosine (0) and HPAA (U) added to pigfeces. (0) No addition.

declined with rising pH values. At pH 5.5, 4-hydroxystyrene was also accumulated (Fig. 3).Aromatic acids. After overnight incubation

of both farm slurry and feces with tyrosine, thegas chromatographical analysis of the acid ex-

tracts showed minor peaks with the same reten-tion time as HPAA, which were absent whentyrosine was not added. However, interpretationof the chromatograms was difficult because ofthe presence of many unknown components.Inoculation of medium A and reinforced clos-tridial medium with 1o-4 g of farm slurry per

liter of medium showed accumulation of mainlyHPPA. Small amounts of HPAA and tyrosolwere also present in both media. A minor peakwith the same retention time as PCA appearedin the gas chromatogram when both trimethyl-silyl and methyl derivatives were prepared. Thispeak could not be identified as PCA with cer-

tainty. Simple phenols were not formed.Radiochemical experiments with farm

slurry. The distribution of radioactivity over

the neutral and acid extracts after incubation of[U-"4C]tyrosine with farm slurry is given in Ta-ble 2. Thin-layer chromatograms of the neu-tral extract showed that radioactivity was pres-

ent only in the spot with the same Rf values as

those of phenol and p-cresol. Gas chromatogra-phy with the use of the effluent splitter con-

firmed that no labeled neutral compounds otherthan phenol and p-cresol were present. In two

slurry samples the ratio of phenol to p-cresolformed was found to approach 1. Aromatic acidspresent in slurry sample I (Table 2) after theincubation period consisted mainly of HPPA(53%) and PPA (25%), with minor quantities of-HPAA and HPLA. In a similar experiment la-beled HPAA and HPPA were incubated withfarm slurry (Table 3). p-Cresol was formed fromHPAA, and no PAA was present, whereas 25%from the added HPPA was recovered dehydrox-ylated as PPA. About 3% of the added label wasfound in the neutral extract.

Table 4 gives the results of a similar experi-ment in which various labeled possible inter-mediates of the anaerobic degradation of tyro-sine were added to farm slurry. In this experi-ment the incubation period was 3 h. No trans-formation products of tyrosol and tyramine weredetected. The transformation patterns of bothHPAA and HPPA corresponded with those inTable 4. Tyrosine and HPLA were obviouslyreadily fermented, tyrosine to mainly phenoland p-cresol, with the intermediate compoundHPAA accumulating in this short-term experi-ment. A minor percentage of the label was lo-

10r

Z- 6

cr4

en2

.4

o

I.

5.5 60 6.5 7.0 7.5 8.0 pH

FIG. 3. Effect ofpH value on the formation of 4-hydroxystyrene (0) and 4-ethylphenol (0) from PCAadded to pig feces.

TABLE 2. Distribution of radioactivity (percent)after addition of L-[U-'4C]-tyrosine to farm slurry"

Waste Phenol + Ratio AromaticpH phenol-p-asample p-cresol cresol acids

I 7.5 51 0.79 12II 7.8 56 11

III 7.7 55 0.84 10IV 7.3 55

a Incubated for 20 h at 20°C.

TABLE 3. Distribution of radioactivity (percent)after addition of[U-'4C1-HPAA or [U-'4C]HPPA to

farm slurrynCom- Aromatic acidpound p-Cresoladded HPAA HPPA PAA PPA

HPAA 75 9 0 0 0HPPA 3 0 71 0 25

n Incubated for 20 h at 20°C.

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TABLE 4. Distribution of radioactivity (percent)after incubation, under anaerobic conditions, of

farn slurry with labeled tyrosine and with labeledpossible internediates of tyrosine degradation'

Phe- Aromatic acidCom- nol Tyro-pound +added p-cre- aol HPLA HPAA HPPA PPA

Sol

Tyrosine 31 0 9 38 6 2Tyrosol 0 100 0 0 0 0Tyramine 0 0 0 tr 0 0HPAA 17 0 0 72 0 0HPPA 3 0 0 0 95 5HPLA 2 0 75 6 11 1

a Incubated for 3 h at 20°C.

cated in HPLA, HPPA, and PPA. The mainproducts formed from HPLA were HPPA andHPAA, though p-cresol and PPA also carriedsome label.Radiochemical experiments with feces.

Labeled tyrosine was added to five samples offreshly voided feces from different pigs. Themixtures were incubated at 370C for 20 h. Anal-ysis of the neutral extract by thin-layer and gaschromatography revealed that it contained no

labeled product other than p-cresol. No labelwas recovered in phenol or 4-ethylphenol. p-

Cresol comprised 19 to 35% (average, 28%) ofthe label added initially. The acid extract con-

tained 35% (range, 31 to 46%) of the addedradioactivity of which 95% (range, 82 to 99%)was present in PPA and 4% (1.5 to 13%) was

present in HPLA; the other aromatic acids wereabsent or present in trace amounts only. In theseexperiments, an average of 63% (71% if includingC02 lost in decarboxylation reactions) of theadded label was recovered.The results of adding labeled tyrosine and

intermediates to feces are given in Table 5. Here,as was the case with fann slurry, no p-cresol or

aromatic acids were produced from either tyro-sol or tyramine. The decarboxylation of HPAAto p-cresol was nearly completed within the 3-hexperimental period. Dehydroxylation ofHPAAwas not obvious but might have occurred in

trace amounts. This is in contrast with HPPA,which was dehydroxylated almost quantitativelyto PPA. p-Cresol and PPA were accumulated inabout equal amounts from the breakdown oftyrosine. The products formed from HPLA were

PPA and p-cresol.

DISCUSSIONLabeled versus unlabeled test com-

pounds. In preliminary experiments, 1 mg of

test compound was added per ml of sample.Addition of compounds in these concentrations

TABLE 5. Distribution of radioactivity (percent)after incubation, under anaerobic conditions, ofpig

feces with labeled tyrosine and labeled possibleintermnediates of tyrosine degradationa

Con- p-Cre- Tyro- Aromatic acid

added o 0 HPLA HPAA HPPA PPA

Tyrosine 38 0 1 2 0 32Tyrosol tr 97 0 0 0 0Tyramine 0 0 0 0 0 0HPAA 73 0 0 9 0 0HPPA 5 0 0 0 7 91HPLA 14 0 40 3 tr 21

a Incubated for 3 h at 37°C.

might lead to erroneous results, due to enzymeinduction or selection of bacteria. These prob-lems could be overcome by the use of radioactivesubstances in combination with short incubationperiods. Comparison of the results obtained withlabeled and nonlabeled test compounds revealedthat they showed the same tendencies. Thus,overnight incubation with 1 mg of test substanceper ml gives good indications as to the processesoccurring in the systems studied.

Phenol. In farm slurry nearly half of thetyrosine is converted to phenol. The involvedbacteria probably possess the enzyme tyrosinephenol-lyase, which degrades tyrosine to phenol,pyruvate, and ammonia (Fig. 4). This conclusionis supported by several observations. Phenol wasreadily produced from tyrosine but only in aminor degree from HPPyrA (Table 1). This ob-servation is consistent with the results obtainedby Ichihara et al. (23) with tyrosine phenol-lyasefrom Escherichia coli. The only other com-pound from which phenol was formed was 4-hydroxybenzoic acid. In farm slurry this com-pound was decarboxylated over the completepH range studied (pH 6.0 to 8.5), whereas phenolwas produced from tyrosine at the higher pHvalues only (pH 7.0 to 8.5). This correspondswith pH ranges reported for tyrosine phenol-lyase from other bacteria (9, 24, 25).A possible explanation for the complete ab-

sence ofphenol production in feces may be foundin the repression of the enzyme tyrosine phenol-lyase by simple carbohydrates which are liber-ated by the degradation of the cellulose-he-micellulose fraction of plant cell wall constitu-ents in feces. In stored farm slurry the moreeasily degradable fraction of plant cell wall ma-terial has already been consumed.p-Cresol. The degradation of tyrosine to p-

cresol via HPPyrA and HPAA is a major path-way in the breakdown of tyrosine in both farmslurry and feces. In experiments with tyrosineadded to the samples, HPAA accumulated (Ta-bles 4 and 5). Pure-culture studies have shown

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IX HO

[' NH2I HO-C-C-COOH

Ct3 0m HO C-C-COOH

HA' ~~~~~0

M HO ¢C-COOH HO o C--C-COOH E

"V'~

HO__C-C-COOH IT

-C_CCOOH Z

FIG. 4. Proposed schemes for the degradation pathways of tyrosine in pig feces (solid arrows) and inanaerobically stored, mixed wastes (dashed arrows). I, Tyrosine; II, HPPyrA; III, HPAA; IV, p-cresol; V,HPLA; VI, PCA; VII, HPPA; VIII, PPA; and IX, phenol.

that many tyrosine-degrading bacteria accumu-late HPAA in their medium, without being ableto decarboxylate this compound (15, 22, 39, 40).From the foregoing it is concluded that theconversion of tyrosine to p-cresol in piggerywastes probably occurs in at least two steps, bydifferent bacteria, the first to HPAA and thesecond to p-cresol.

4-Hydroxybenzyl alcohol and 4-hydroxy-benzaldehyde were found to be reduced mainlyto p-cresol in feces and in slurry, with minoramounts of phenol being formed (Table 1).Phenol is obviously formed via oxidation to 4-hydroxybenzoic acid followed by decarboxyla-tion. Similar results have been obtained by Sche-line (35) with rat intestinal contents. Whetherthese transformations are part of the tyrosinemetabolism is not clear. Japanese workers foundaccumulation of 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid when Proteus vulgaris wasincubated with tyrosine (21, 40). However, themethods applied by these authors to isolate 4-hydroxybenzaldehyde from the incubation mix-ture may not have excluded the formation ofthis compound by chemical decompostion ofpossibly accumulated HPPyrA (8).When feces or slurry was incubated with ra-

dioactive HPPA, 2 to 5% of the label was re-covered in the neutral extract. Theoretically,phenol may be produced from HPPA by ,f-oxi-dation to p-hydroxybenzoic acid and successivedecarboxylation. However, longer incubation pe-

riods with labeled HPPA did not result in higherrecoveries of label in the neutral extract indicat-ing that artefacts may have been involved. Theresults reported in Table 5 indicate that theconversion of HPPA to PPA in feces proceedsmuch more readily than in farm slurry. In con-trast to feces, the pathway of reductive tyrosinedegradation (from HPPyrA -- HPPA; Fig. 4) isof minor importance in farm slurry.Tyrosine degradation with decarboxylation to

tyramine as the first step and continuing accord-ing to the scheme tyramine -+ tyrosol -- HPPA-+ p-cresol could not be demonstrated in eitherfarm slurry or feces (Tables 4 and 5). That thisis a possible route is concluded from experimentswith long incubation periods with tyramine andtyrosol (Table 1). In these long-term experi-ments large amounts of p-cresol were producedfrom both substances. Tyramine has been re-ported to be produced in small amounts by themicroflora ofhuman feces. Asatoor (1) recovered0.6% of the labeld tyrosine added to human fecalbacteria as tyramine after 25 h at 35°C.Reductive degradation of tyrosine. PCA

is probably the intermediate in the reduction ofHPLA to HPPA. However, this compound hasnot been shown to be present in the wastes.Tanaka (41) described this pathway for P. vul-garis, and earlier Hirai (20) had noticed theaccumulation of large amounts of PCA by thesame organism in a medium with tyrosine. Inaddition, the formation of the corresponding

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ANAEROBIC DEGRADATION OF TYROSINE 637

acrylic acids from both indolelactic (18, 30) andphenyllactic (18, 29) acids has been reported tooccur in the degradation of tryptophan andphenylalanine, which are structurally related totyrosine. The occurrence of PCA as an inter-mediate in the decomposition of tyrosine toHPPA makes it likely that 4-ethylphenol canalso be a product of tyrosine metabolism. PCAis readily converted to 4-ethylphenol by feces(Table 1), which agrees with the findings ofScheline (34) and Bakke (2). The accumulationof 4-hydroxystyrene in feces incubated at pH 5.5(Fig. 3) and the observation that 4-ethylphenolis not produced from HPPA (Table 1; 2, 12, 34)suggest that PCA is first decarboxylated to 4-hydroxystyrene, followed by hydrogenation to 4-ethylphenol. Decarboxylation of PCA in pureculture has been reported for an Aerobacter sp.(17), a Bacillus sp. (24), and a number of fungi(7, 19). The conversion of PCA to 4-ethylphenolvia hydrogenation to HPPA and decarboxyla-tion, as proposed first by Baumann (6) and laterby other authors (13, 42), is not likely to occurin the wastes studied here.Decarboxylation reactions. A remarkable

process in farm slurry as well as in feces is thedecarboxylation of various (4-hydroxyphenyl)-carboxylic acids (Table 1; Fig. 4). These decar-boxylations did not occur when synthetic mediawere inoculated with farm slurry (see Results).In these experiments HPPA and HPAA accu-mulated. With respect to the organisms andenzymes involved in the decarboxylation reac-tions, not much is known. Scheline (33) con-cluded that decarboxylation of aromatic acidsoccurs only when a free hydroxyl group is pres-ent in the para-position. Finkle et al. (17) re-ported a constitutive enzyme which decarbox-ylates PCA; this enzyme also decarboxylatedother phenylacrylic compounds bearing a 4-hy-droxy group in the ring. The same bacteriumcould form a different adaptive enzyme able todecarboxylate 4-hydroxybenzoic acid, but notHPAA, so probably different enzymes decarbox-ylate PCA, HPAA, and 4-hydroxybenzoic acid.Dehydroxylation of HPPA. In feces, PPA

is a main degradation product of tyrosine,whereas in farm slurry only minor amounts ofthis compound are formed (Tables 3 and 5). Thedehydroxylation of HPPA to PPA has been re-ported to occur also in sheep rumen (36) and inhuman feces (12); it may be a common reactionin anaerobic environments. The significance ofthe dehydroxylation of HPPA may lie in thelinkage of the degradative pathways of tyrosineand phenylalanine.

Curtius et al. (12) reported the occurrence ofrearrangement reactions of ring substituents

leading to the formation of trace amounts of 3-hydroxyphenyl compounds. These reactionshave not been considered in the work reportedhere.

ACKNOWLEDGMENTSThanks are due to the Insitute for Animal Nutrition Re-

search in Wageningen and to the Research Institute for Ani-mal Feeding and Nutrition "Hoorn" in Lelystad for the pro-vision of pig feces. This study was financed by the CommissieHinderpreventie Veeteeltbedrijven.

LITERATURE CIMD1. Asatoor, A. M. 1968. The origin of urinary tyramine.

Formation in tissues and by intestinal microorganisms.Clin. Chim. Acta 22:223-229.

2. Bakke, 0. M. 1969. Studies on the degradation of tyrosineby rat caecal contents. Scand. J. Gastroenterol.4:603-608.

3. Bakke, 0. M. 1969. The effect of neomycin and experi-mental coprostasis on the excretion of simple phenolsin the rat. Scand. J. Gastroenterol. 4:419-423.

4. Bakke, 0. M. 1969. Urinary simple phenols in rats fedpurified and nonpurified diets. J. Nutr. 98:209-216.

5. Bakke, 0. M. 1969. Urinary simple phenols in rats feddiets containing different amounts of casein and 10%tyrosine. J. Nutr. 98:217-221.

6. Baumann, E. 1879. Ueber die Bildung von Hydropara-cumarsaure aus Tyrosin. Ber. Dtsch. Chem. Ges.12:1450-1454.

7. Bayne, H. G., B. J. Finkle, and R. E. Lundin. 1976.Decarboxylative conversion of hydroxycinnamic acidsto hydroxystyrenes by Polyporus circinata. J. Gen.Microbiol. 95:188-190.

8. Billek, G. 1963. p-Hydroxyphenylpyruvic acid. Org.Synth. 43:49-54.

9. Brot, N., S. Smit, and H. Weissbach. 1965. Conversionof L-tyrosine to phenol by Clostridium tetanomorphum.Arch. Biochem. Biophys. 112:1-6.

10. Carman, G. M., and R. E. Levin. 1977. Partial purifica-tion and some properties of tyrosine phenol lyase fromAeromonas phenologenes ATCC 29063. Appl. Environ.Microbiol. 33:192-198.

11. Chen, T. C., and R. E. Levin. 1975. Isolation of Aero-monas sp. ATCC 29063, a phenol-producing organism,from fresh haddock. Appl. Microbiol. 30:120-122.

12. Curtius, H. C., M. Mettler, and L. Ettlinger. 1976.Study of the intestinal tyrosine metabolism using stableisotopes and gas chromatography-mass spectrometry.J. Chromatogr. 126:569-580.

13. Drasar, B. S., and M. J. Hill. 1974. Human intestinalflora. Academic Press Inc., New York.

14. Ehrlich, F. 1911. Uber die Vergarung des Tyrosins zu p-Oxyphenylathylalkohol (Tyrosol). Ber. Dtsch. Chem.Ges. 44:139-146.

15. Elsden, S. R., M. G. Hilton, and J. M. Waller. 1976.The end products of the metabolism of aromatic aminoacids by Clostridia. Arch. Microbiol. 107:283-288.

16. Enei, H., H. Matsui, K. Yamashita, S. Okumura, andH. Yamada. 1972. Distribution of tyrosine phenol lyasein microorganisms. Agric. Biol. Chem. 36:1861-1868.

17. Finkle, B. J., J. C. Lewis, J. W. Corse, and R. E.Lundin. 1962. Enzyme reactions with phenolic com-pounds: formation of hydroxystyrenes through the de-carboxylation of 4-hydroxycinnamic acids by Aerobac-ter. J. Biol. Chem. 237:2926-2931.

18. Hansen, L. L., and M. A. Crawford. 1968. Bacterialdegradation of the aromatic amino acid side chain.Biochem. Pharmacol. 17:338-342.

19. Harada, T., and Y. Mino. 1976. Some properties of p-

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coumarate decarboxylase from Cladosporium phlei.Can. J. Microbiol. 22:1258-1262.

20. Hirai, K. 1921. Uber die Bildung von p-Oxyphenylessig-saure und p-Oxyphenylacrylsaure aus L-Tyrosin durchBakterien. Biochem. Z. 114:71-80.

21. Hirai, K. 1923. Uber das Vorkommen von p-Oxybenzal-dehyd und p-Oxybenzoesaure bei der bakteriellen Ty-rosinzersetzung, mit besonderer Beriicksichtigung desdabei gebildeten Melanins. Biochem. Z. 135:299-307.

22. Hirai, K., and T. Tanaka. 1963. Decomposition of L-tyrosine by Bac. Coli (5). Paper chromatographicaldetection of decomposition products of L-tyrosine byBac. Coli. Bull. Pharm. Res. Inst. (Osaka) 45:1-10.

23. Ichihara, K., H. Yoshimatsu, and Y. Sakamota. 1956.Studies on phenol formation. III. Ammonium and po-

tassium ions as the activator of beta-tyrosinase. J. Bio-chem. 43:803-810.

24. Indahl, S. R., and R. R. Scheline. 1968. Decarboxylationof 4-hydroxycinnamic acids by Bacillus strains isolatedfrom rat intestine. Appl. Microbiol. 16:667.

25. Kumagai, H., N. Kashima, H. Torii, H. Yamada, H.Enei, and S. Okumura. 1972. Purification, crystalli-zation and properties of tyrosine phenol lyase fromErwinia herbicola. Agric. Biol. Chem. 36:472-482.

26. Kumagai, H., H. Matsui, and H. Yamada. 1970. For-mation of tyrosine phenol-lyase by bacteria. Agric. Biol.Chem. 34:1259-1261.

27. Kumagai, H., H. Yamada, H. Matsui, H. Ohkishi, andK. Ogata. 1970. Tyrosine phenol lyase. I. Purification,crystallization and properties. J. Biol. Chem. 245:1767-1772.

28. Lederer, M. E. 1943. Isolement de p-ethyl-phenol a partirde l'urine de jument gravide. Trav. Membres Soc. Chim.Biol. 25:1237-1238.

29. Moss, C. W., M. A. Lambert, and D. J. Goldsmith.1970. Production of hydrocinnamic acid by clostridia.Appl. Microbiol. 19:375-378.

30. O'Neil, S. R., and R. D. DeMoss. 1968. Tryptophantransaminase from Clostridium sporogenes. Arch. Bio-chem. Biophys. 127:361-369.

31. Prevot, A. R., and R. Saissac. 1947. Recherches sur laproduction des phenols par les bacteries anaerobies.Ann. Inst. Pasteur Paris 73:1125-1129.

32. Schaefer, J. 1977. Sampling, characterisation and analy-sis of malodours. Agric. Environ. 3:122-127.

33. Scheline, R. R. 1966. Decarboxylation and demethylationof some phenolic benzoic acid derivatives by rat caecalcontents. J. Pharm. Pharmacol. 18:664-669.

34. Scheline, R. R. 1968. Metabolism of phenolic acids by therat intestinal microflora. Acta Pharmacol. Toxicol.26:189-205.

35. Scheline, R. R. 1972. The metabolism of some aromaticaldehydes and alcohols by the rat intestinal microflora.Xenobiotica 2:227-236.

36. Scott, T. W., P. F. V. Ward, and R. M. C. Dawson.1964. The formation and metabolism of phenyl-substi-tuted fatty acids in the ruminant. Biochem. J. 90:12-24.

37. Spoelstra, S. F. 1977. Simple phenols and indoles inanaerobically stored piggery wastes. J. Sci. Food Agric.28:415-423.

38. Stahl, E. 1967. Dunnschichtchromatographie. Springer-Verlag, Berlin.

39. Stone, R. W., H. E. Machamer, W. J. McAleer, and T.S. Oakwood. 1949. Fermentation of tyrosine by marinebacteria. Arch. Biochem. 21:217-223.

40. Tanaka, T. 1965. The decomposition of L-tyrosine and itsderivatives by Proteus vulgaris (2). Production of p-hydroxyphenylacetic acid p-hydroxybenzaldehyde andmelanin from L-tyrosine. Bull. Pharm. Res. Inst.(Osaka) 59:1-10.

41. Tanaka, T. 1968. The decomposition of L-tyrosine and itsderivatives by Proteus vulgaris (4). The decompositionof p-hydroxyphenyllactic acid, p-hydroxyphenylacrylicacid and p-hydroxyphenylpropionic acid. Bull. Pharm.Res. Inst. (Osaka) 74:1-10.

42. Van der Heiden, C., S. K. Wadman, D. Ketting, andP. K. de Bree. 1971. Urinary and faecal excretion ofmetabolites of tyrosine and phenylalanine in a patientwith cystic fibrosis and severely impaired amino acidabsorption. Clin. Chim. Acta 31:133-141.

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