Metabolism of nitrogen compounds by hydrogenomonas eutropha

BIOCHIMICA ET BIOPHYSICA ACTA BBA

I35

25761

METABOLISM OF N I T R O G E N COMPOUNDS BY

HYDROGENOMONAS EUTROPHA I. U T I L I Z A T I O N OF U R I C ACID, ALLANTOIN, H I P P U R I C ACID, AND C R E A T I N I N E ELIZABETH C. B. AMMANN AND LAWRENCE L. REED Lockheed Missiles and Space Company Research Laboratories, Palo Alto, Calif. (U.S.A.) (Received November i7th, 1966)

SUMMARY I. AutotrophicaUy grown Hydrogenomonas eutropha can utilize uric acid, allantoin, hippuric acid, and urea as sole nitrogen sources for growth when energy is available from the oxidation of H , gas. 2. The initial degradation of uric acid, allantoin, and urea occurs b y oxidative and hydrolytic mechanisms in which hydrogen is not required. The decomposition of uric acid and allantoin proceeds at the same rate in either a H~ or H~-free gas environment, while the degradation of urea is slowed down considerably in the latter. Urea and N H 3 are intermediates of these reactions. H2 is required for the incorporation of the N H 3 into cell material. 3. The initial degradation of hippuric acid also occurs without the necessity of a source of H 2. The decomposition rate is equal in either gas environment. Glycine appears to be an intermediate of this reaction. 4. Uric acid, aUantoin, and hippuric acid can be used for cell growth in the absence of externally supplied CO s when energy is available from the oxidation of H,. Urea is only utilized to a small extent under these environmental conditions. 5. Creatinine is neither utilized or degraded b y H. eutropha in any of the gas environments employed. 6. The ability of H. eutropha to utilize a widevariety of man's nitrogenous waste products supports the use of this organism in a bioregenerative life support system.

INTRODUCTION The bacterium Hydrogenomonas eutropha is a facultative autotroph which can use energy obtained from the oxidation of H , to form cell material from CO 2 and inorganic salts. The capacity of this organism to utilize organic nitrogen compounds under these conditions has not been analyzed extensively to date. REPASKE1 has reported that this bacterium grows well on urea in a H2, 02, CO s gas environment. Wild-type Hydrogenomonas facilis has been found to utilize either phenylalanine or tyrosine for growth with glucose as the main carbon and energy source 2. A study of the ability of H. eutropha to utilize allantoin, creatinine, hippuric acid, uric acid,

BiocMm. Biophys. Acta, i41 (1967) 135-i43

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E.c.B.

AMMANN, L. L. R E E D

and urea under autotrophic growth conditions was undertaken in the present series of experiments. MATERIALS AND METHODS

Chemicals Uric acid and creatinine were obtained from Eastman Organic Chemical Company, urea and NH4C1 from Mallinckrodt Chemical Works, hippuric acid from Matheson Company, and allantoin from Nutritional Biochemical Corporation. The purity of these compounds was checked by melting point and infrared analyses. Culture organism and media H. eutropha was obtained from the culture collection of D. Davis, Department of Bacteriology, University of California, Berkeley. TABLE

I

BASAL CULTURE MEDIUM USED TO GROW H. eulropha (0.2 M PHOSPHATE BUFFER)

Component

Concn. (g/l)

K2HPO4.3H20 KH2PO 4 CaCI~. 2 H 2 0 NaC1 M g S O 4 • 7 H..,O FeC13" 6 H 2 0 CoC12. 6 H 2 0 C u S O 4. 5 H 2 0 Z n S O 4. 7 H 2 0 MnC12" 4H2 O N a 2 M o O 4. 2 H 2 0 HsBOa

22.8 13.6 o.i o. i o. i 0.05 2" lO -5 2" lO -4 I" lO -3 4" I ° - ~ I . lO -3 3" 1 ° - 4

The basal culture medium contained o.2 M or o.oo2 M phosphate buffer (pH 6.7) (Table I). The more concentrated buffer was used in the growth studies while the lower concentration was employed in the degradation experiments. AUantoin, creatinine, hippuric acid, urea, uric acid, and NI-I4C1 were added individually to the basal medium to given the carbon and nitrogen concentrations shown in Tables II and III. The pH did not change with the addition and utilization of the nitrogen compounds when the high phosphate buffer was used. All media were sterilized by filtration through Pyrex ultrafine sintered-glass filters (pore diameter o.9-1.4/, ). The iron solution was filtered separately and added after the rest of the components had been sterilized.

Gas environment Three different gas environments were employed: (a) H~-O~-C02 (70: 20: IO, v/v), (b) I-I2-02-N ~ (70: 20: IO, v/v), and (c) N~-02-CO z (70:20: io, v/v). These gases were mixed and maintained at atmospheric pressure by a water-displacing system consisting of two 19-1 Pyrex bottles connected by 8 mm diameter glass tubing. This system is similar to that of REPASKE1. The gases were mixed by individually introBiochim. Biophys. Aaa, 141 (1967) i 3 5 - x 4 3

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M E T A B O L I S M OF N I T R O G E N C O M P O U N D S BY H Y D R O G E N O M O N A S TABLE

II

CONCENTRATION OF NITROGEN COMPOUNDS ADDED TO BASAL CULTURE MEDIUM FOR GROWTH STUDIES

Component

Allantoin Creatinine Hippuric acid Urea Uric acid NH4C1

TABLE

H. eutropha

Concn. (rag~l) Component

Nitrogen

Carbon

652 47o 33o 990 555 380

23 ° 175 26 462 185 IOO

2oo 2oo 2oo 200 2oo --

III

CONCENTRATION OF NITROGEN COMPOUNDS ADDED TO BASAL CULTURE MEDIUM FOR H . e$~tropha DEGRADATION STUDIES

Component

Allantoin Creatinine Hippuric acid Urea Uric acid NH4C1 NH4C1

Cohen. (rag/l) Component

Nitrogen

Carbon

283 27 ° 33 ° 214 75 382 96

ioo i oo 26 IOO 25 IOO 25

86 I 15 199 43 27 ---

ducing pure H,, 0 2, CO, or N, into the first bottle filled with water, thereby displacing the proper volume of liquid into a second bottle. The final gas volume was 8 1. Each mixture was introduced into the culture flasks after air had been evacuated. Water displaced back into the first bottle as gas utilization occurred, maintaining one atmosphere pressure throughout the experiments.

Culture apparatus The culture apparatus consisted of eight 25o-ml erlenmeyer flasks, each fitted with a side-arm 3 cm in length and located 7-5 cm from the bottom of the flask. Sampling was accomplished by inserting a syringe through a protected rubber stopper on the side-arm. The flasks were connected by ground joints and vacuum tubing to a manifold and the gas-water reservoir bottles. Agitation and temperature control (30-32 °) were obtained by placing the flasks in a New Brunswick Scientific C. water-bath shaker (Model G77 ). The entire culture unit was placed in a hood for safety. Growth studies Nitrogen utilization was determined by measuring the growth of H. eutropha over a time interval of 2-3 days. A5-ml H. eutropha inoculum, which had been washed after being grown up on NH4C1, was added to each of 7 culture flasks containing Biochim. Biophys. Acta,

141 (1967) 1 3 5 - i 4 3

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E . C . B . AMMANN, L. L. REED

5o ml nutrient media consisting of the 6 different nitrogen compounds and a nitrogenfree control. Cell count equalled 3" lO8-4 . lOS cells per ml immediately after inoculation. 5-ml samples were withdrawn from each flask over the following 48-h interval for pH, growth, and contamination ana:yses. Contamination was checked by plating the cultures on both heterotrophic medium (5 g yeast extract, I g K2HPO 4. 3H20, 0.5 g MgSO4" 7H20 per 1 distilled water) and autotrophic medium (Table I with 2 mM phosphate buffer (pH 6.8) and 7 mM NH,C1) and incubating the plates in the proper gas environment at 3 o°. Growth was measured by determining the absorbance of the culture medium at 650 nm and by counting the cells on both the heterotrophic and autotrophic plates. Growth studies were carried out in all three gas environments.

Degradation studies The degradation of the nitrogen compounds by H. eutropha was studied by a procedure that was similar to the one described for determining growth. In addition to the pH, cell count, and contamination measurements, 5-ml samples were withdrawn to determine the concentration of nitrogen compounds found in the nutrient medium. The cells were removed by centrifugation at 5000 rev./min. Uric acid, hippuric acid, creatinine, and allantoin concentrations were measured by ultraviolet light absorption from 360 to 21o nm with a Beckman DK2A spectrophotometer. Absorption maxima are present at 29° n m for uric acid, 223 nm for hippuric acid, and 229 nm for creatinine at pH 6. 7. The increasing absorption of ultraviolet light by allantoin in the lower wavelength range was measured at 230 nm. Urea and ammonia were analyzed by colorimetry using Nessler's reagent 3. Light absorption was measured at 505 nm with a Bausch and Lomb Spectronic 20 colorimeter. All nitrogen compounds were also detected by paper chromatography. RF values were determined on descending chromatograms. The solvent was n-butanolglacial acetic acid-water (58:13:29, v/v). Detection methods included Ehrlich's reagent 4, an ultraviolet lamp (Ultraviolet Products, Inc., Model 5-81, 253 nm transmission maximum), and a mercuric acetate-diphenyl carbazone spray test 5. Several concentrations of each sample were run. RESULTS

Growth of H. eutropha on various nitrogen compounds H. eutropha can utilize allantoin, hippuric acid, urea, and uric acid as sole nitrogen sources for growth in an atmosphere containing H 2, 02, and CO 2. Comparatively good rates of growth are obtained on these organic nitrogen compounds in relation to NH4C1 (Fig. ia). The rate of growth on hippuric acid, although lower in the 0.2 M phosphate buffer, is equal to the other nitrogen compounds in a 2 mM phosphate buffer (Fig. 2). Perhaps, the high phosphate concentration adversely affects an enzyme or intermediate of hippuric acid metabolism. Energy must be available from the oxidation of H 2 in order for H. eutropha to utilize the above nitrogen compounds under these autotrophic growth conditions (Figs. i a and Ib). Growth of the bacterium does not occur on allantoin, hippuric acid, urea, uric acid or ammonium chloride in the absence of this gas. Externally supplied CO, is not essential for the utilization of allantoin, hippuric acid, and uric acid by H. eutropha (Fig. IC). A 5- to io-fold increase in cell Biochim. Biophys. Acta, 141 (1967) 135-143

METABOLISM

OF NITROGEN

COMPOUNDS

139

BY HYDROGENOMONAS

concentration occurs when CO s has been replaced by N~, as long as energy is available from the oxidation of H 2. The rate of growth in either gas environment is equivalent as seen by a comparison of the slopes of Figs. Ia and IC. Growth stops sooner in the allantoin and uric acid media when CO~ has not been added even though the pH has not changed. Perhaps the carbon of the compound has been completely utilized, is no longer present in an available form, or has escaped to the 8-1 atmosphere after being degraded to CO~. The inclusion of IO % CO~ causes growth to resume again. (a)

(b)

URICACID ALLANTOIN~ UREA/~

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, / /4/ t AMMON'U" C.LOR,OE / f / ~UREA

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.... 70% He: 20%02: 0% CO2 ADDED

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(c)

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"~Ff" .........................CREATININE ......

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NITROGENFREE

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CREATIN I N E AND N ITROGEN-FREE

,~'o 5'0 io

7oo

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TIME (H)

;o

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F i g . I. G r o w t h of H. eutropha on v a r i o u s n i t r o g e n c o m p o u n d s in t h e presence of different gas e n v i r o n m e n t s : (a) H ~ - O ~ - C O 2 ( 7 0 : 2 0 : IO, v / v ) (b) N ~ - O 2 - C O 2 ( 7 o : 2 o : I o , v / v ) (c) H , , - O 2 - N ~ (7 ° : 2 0 : IO, v / v ) .

1.0 0.8

0.7 0.6 0.5 o

t.O 'qt z

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~

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F

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0.4 0.5

FER

_0 P< 0.2 E Z

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~ 0.08'.

I IO

I 20

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I 50

f 40

50

TIME (H) F i g . 2. G r o w t h of H. eutropha on hippuric acid in m e d i a c o n t a i n i n g different c o n c e n t r a t i o n s of p h o s p h a t e buffer. The gas a t m o s p h e r e w a s H ~ - O 2 - C O ~ ( 7 o : 2 o : 1 o , v / v ) .

The utilization of hippuric acid by H. eutropha in either the presence or absence of CO 2 is identical. This compound must supply all the necessary carbon for the production of cell material. It is not known whether the carbon of these compounds is used only after being degraded to CO s or whether other, larger carbon-containing molecular fragments can be incorporated into cell material. Biochim. Biophys. Acta, 1 4 1 ( 1 9 6 7 ) 1 3 5 - 1 4 3

140

E . C . B . AMMANN, L. L. REED

I t is not possible, from the growth studies illustrated in Fig. IC, to determine whether H. eutr@ha can grow on urea in the absence of externally supplied CO 2. In a high phosphate buffer, growth on NH4C1 increases at the same slow rate as on urea, suggesting that the cells of both flasks m a y be obtaining CO s released from the breakdown of uric acid, allantoin, or hippuric acid. However, in a low phosphate buffer, growth on urea is greater than on NH4C1 (Fig. 3), indicating that the organism m a y use urea to a small extent in the absence of externally supplied CO s. H. eutropha does not grow on creatinine in any of the gas environments employed (Figs. Ia, Ib, and IC). 1.0 0.8 0.7 O.6 0.5

0.4 UREA

~

0.2

E

I--

llg I.z~

~

0.1

o 0.08

~

I

I0

om

"-AMMONIUM

~m~.~.~'~

CHLORIDE I

I

I

20 30 TIME (H)

40

50

Fig. 3. G r o w t h of H. e~tropha on urea in the absence of externally supplied CO 2.

Degradation of nitrogen compounds by H. eutr@ha At p H 6.2, whole cells of H. eutropha degrade allantoin, hippuric acid, urea, uric acid, and NH,C1 at different rates in a complete gas atmosphere (Fig. 4a). A1lantoin is utilized the fastest at a rate of 6.3 mg/h. Uric acid and hippuric acid degradation is approximately equal, the average rate being 2.5 mg/h. Urea and NH4CI are utilized more slowly with values of 1.o-1.5 mg/h. These latter two compounds have lag times from 5 to IO h before breakdown begins. Creatinine is not degraded throughout the entire 49-h time interval. The omission of either H 2 or CO s does not interfere with the degradation of

~4c

~ zo

(o) ~IPPURICACID

(c}

(b)

" om~ / ~ \ ' ~ REA OnnI_ 1C :,~L~ ~-~"--~ALLANTOIN l- Z '~ ~h. ~ P ~ U R I C ACID ZbJ 0 ' ~ 7 - ~ I ~ N H ~ 'C[ , , Z 0 o 10 20 30 40 o

NH~.CI

= NH4CI

CREATININE~ " TOIN

~..~¢~uRic ACID 50

'~. ~,\HIPPURIC ACID, , ~ 10 20 30 40 TIME (H)

50

10

20

30

40

50

Fig. 4. D e g r a d a t i o n of various nitrogen c o m p o u n d s b y H. eutropha in the presence of different gas e n v i r o n m e n t s : (a) Hz-O2--CO 2 (7 ° : 20 : IO, v/v) ; (b) N~-Oz-COa (7 ° : 20 : IO, v/v) a n d (c) H2-O2-N 2 (70:20: lO, v/v).

Biochim. Biophys. Acta, 141 (1967) 135-143

141

METABOLISM OF NITROGEN COMPOUNDS BY HYDROGENOMONAS

uric acid, allantoin, and hippuric acid (Figs. 4b and 4c). The disappearance of these compounds from the nutrient medium proceeds at the same rate in all the gas environments employed. However, urea degradation is slowed down considerably when either CO 2 or H , is absent, the rate being approx. 0.5 mg/h. NH4C1, like creatinine, is not used in a CO,-free or H~-free atmosphere. Shortly after the addition of H. eutro~ha to culture medium containing uric acid and allantoin, the intermediate compounds, urea and NH4C1, are detected in all the gas environments employed (Figs. 5 a, 5b and 5c). The nutrient medium is devoid of both the original nitrogen compound and the intermediates after 18-25 h in a complete gas environment. The cell concentration has increased approx. 5- to 8-fold by this time and continues to increase to Io-fold its original concentration in the next 25-30 h (Fig. 5). In a gas environment devoid of H 2 or COz, both urea and NH4C1 remain in the nutrient medium for longer time intervals. Urea is gone after 18-25 h in the uric acid flask. It reaches low levels after approx. 30 h in the allantoin flask but traces still remain at 48 h. N H 3 continues to accumulate in the culture medium in the absence of a complete gas environment. When CO~ is absent, N H z builds up to I mg per IOO ml in the uric acid flask and 4 mg per IOO ml in the allantoin one. When H~ is removed, N H s accumulates in larger quantities. Approx. 2 mg per IOO ml is found in the uric acid flask and 6 mg per IOO ml in the allantoin one. A1lantoin is not detected in the nutrient medium of uric acid-grown cells in any of the gas environments employed. N H 3 is detected as an intermediate of urea metabolism by H. eutrojbha. In a complete gas atmosphere the two compounds are gone from the nutrient medium in 18 h and the cells have increased approx. 4-fold. Cell growth continues for another 20 h to a m a x i m u m increase of over Io-fold. When H2 or CO~ are omitted from the

URICACID ~ *UREA

(a) ~Z5

7.5

(b)

(c)

I

*AMMONIA ALLANTOIN

[

*UREA *AMMONIA

I

UREA *AMMONIA

AMMONIUM CHLORIDE CREATININE

"1

HIPPURICACID *GLYCINE

M

*AMMONIA

lb *INTERMEDIATES

20

250

~,0

10 ~) ~0 =~0 EXPERIMENT DURATION(H)

10

20

~

'40

Fig. 5. D e t e c t i o n of i n t e r m e d i a t e s d u r i n g t h e d e g r a d a t i o n of v a r i o u s n i t r o g e n c o m p o u n d s b y H. eutropha in t h e p r e s e n c e of different g a s e n v i r o n m e n t s : (a) H 2 - O 2 - C O z (70:20: io, v/v); (b) N 2 - O 2 - C O 2 (7o: 2o: io, v / v ) ; a n d (c) H 2 - O z - N 2 (7o: 20: IO, v/v). T h e n u m b e r s w i t h i n t h e b a r s r e p r e s e n t t h e c o n c e n t r a t i o n of e a c h c o m p o u n d a t its m a x i m u m level in m g per IOO ml. T h e t i m e i n t e r v a l d u r i n g w h i c h t h e m a x i m u m c o n c e n t r a t i o n s of t h e c o m p o u n d s were o b s e r v e d is r e p r e s e n t e d b y t h e black p o r t i o n of e a c h bar.

BioGhim. Biophys. Acta, 141 (1967) 1 3 5 - I 4 3

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E. C. B. AMMANN, L. L. REED

gas atmosphere, urea is degraded much more slowly. Some urea is still present at the end of 48 h. During this time interval, N1.1~ has increased in concentration. At 48 h, 5 mg of NH3 nitrogen is present per IOO ml of nutrient medium. Neither glycine or N H 3 are detected as intermediate compounds when H. eutropha is grown on hippuric acid in a complete gas environment or when CO 2 has been omitted. However, when I-12 is omitted, a small amount of glycine (I mg per IOO ml) is observed for a short time interval. Traces of N H 3 also accumulate and by the end of the experiment are present in the amount of 0.5 mg per IOO ml of nutrient. DISCUSSION

Pathway of incorporation It appears that H. eutropha initially cleaves uric acid, allantoin, urea, and hippuric acid by oxidative and hydrolytic mechanisms similar to those described for m a n y organisms, including bacteria 6-s, yeast 9, and algae 1°. Degradation of these four compounds occurs in the absence of H2. Well-known intermediates are detected; urea and NHH~ accumulate in the nutrient medium containing uric acid and allantoin, NH~ accumulates in the urea cultures, and glycine is observed for a short time with hippuric acid. Allantoin, a normal metabolic intermediate in the oxidation of uric acid, was not observed in the present study. I t is probable that the compound does not accumulate in the culture medium since it was found to be degraded faster than uric acid. Urea and NHHawould naturally accumulate since these two compounds were found to be incorporated much more slowly than either uric acid or allantoin. Another possibility is that the initial breakdown of uric acid occurs b y a different mechanism which does not yield allantoin. The complete sequence of reactions in the degradation of these nitrogen compounds by H. eutropha has not been determined. One difference in the metabolism of uric acid, allantoin, urea, and hippuric acid by H. eutropha, from the growth of other organisms, is that the energy for the utilization of these compounds can be obtained from the oxidation of HH~.The point in degradation at which this energy source becomes essential for further metabolism resides at the N H a level for uric acid, allantoin, and urea (at least in part). NH~ accumulates in the nutrient medium containing uric acid, allantoin, and urea when H 2 is absent from the gas environment; cell growth does not occur. Almost all of the uric acid-nitrogen is present as NHH3 by the end of the experiment. Approx. 4 mg per I00 ml of allantoin-nitrogen and 5 mg per i00 ml of urea-nitrogen are not accounted for in N H 3. I t is conceivable that this N H 3 has entered the cell without being utilized. I t is also possible that another nitrogen-containing intermediate, which requires an energy source for incorporation into cell material, accumulates and has not been detected. The present series of experiments do not determine at what point energy is required for hippuric acid incorporation. Degradation can proceed beyond glycine without requiring a source of H 2. This intermediate compound appears for only a short time in the nutrient medium at the beginning of the experiment. I t is not known what degradation products accumulate and whether they are located in the nutrient medium or within the cells. A spot which reacts slightly with the ninhydrin spray test and which moves very slowly in the n-butanol-glacial acetic acid-water solvent was seen to accumulate in the culture medium throughout the experiment. Only Biochim. Biophys. Acta, 141 (I967) i 3 5 - I 4 3

METABOLISM OF NITROGEN COMPOUNDS BY HYDROGENOMONAS

I43

0.5 mg per IOO ml of hippuric acid-nitrogen is found as NH 8, which is roughly one-fifth of the nitrogen present in the original compound.

Bioregenerative life support H. eutropha is being considered as a component of a bioregenerative life support system to sustain man in space for long periods of timelk The organism would be used to reduce C0 2 to cell material and to return a portion of man's other waste products to the enclosed cycle. The above experiments support the use of Hydrogenomonas for waste treatment. The organism is capable of degrading and utilizing uric acid, allantoin, hippuric acid, and urea for growth. These compounds are important nitrogen-containing products excreted by man. The bacterium is more versatile with regard to nitrogen metabolism than other organisms being considered for bioregenerative life support systems. Both Hydrogenomonas and Chlorella, the unicellular alga, can use uric acid and urea as sole nitrogen sources for growth 1., but allantoin and hippuric acid are only metabolized by the former. Neither organism utilizes creatinine, which would accumulate in recycled medium. REFERENCES i R. REPASKE, f . Bacteriol., 83 (1962) 418. 2 B. T. DECIcco AND W. W. UMBREIT, J. Bacteriol., 88 (1964) 159o. 3 W. W. UMBREIT, R. H. BURRIS AND J. F. STAUFFER, Manometric Techniques, 3rd ed., Burgess, Minneapolis, Minnesota, 1959, p. 238. 4 E. G. BOLLARD, Australian J. Biol. Sci., IO (1957) 293. 5 E. C. B. AMMANN AND V. H. LYNCH, Anal. Biochem., 7 (1964) 387 • 6 E. G. YOUNG AND W. W. HAWKINS, J. Bacteriol., 47 (1944) 351. 7 W. FRANKE AND G. E. HAHN, Z. Physiol. Chem., 299 (1955) 15. 8 L. L. CAMPBELL, Jr., Biochim. Biophys. Acta, 18 (1955) 16o. 9 F. J. DICARLO, A. S. SCHULTZ AND A. M. KENT, Arch. Biochem. Biophys., 44 (1953) 468. i o E. C. B. AMMANN AND V. H. LYNCH, Biochim. Biophys. Acta, 87 (1964) 37 o. I I L. BONGERS AND B. KOK, Develop. lnd. Microbiol., 5 (1964) 183. 12 E. C, BIRDSEY AND V. H. LYNCH, Science, 137 (1962) 763 .

Biochim. Biophys. Acta, 141 (x967) 135-143