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Inhibition of the fermentation process in soil by acetylene
Soil Bid. Biochem. Vol. 24, No. 9, pp. 905-91 I, 1992 Printed in Great Britain. Ail rights reserved
003&0717/92 SS.00 + 0.00 Copyright 0 1992 Pergamon Press Ltd
INHIBITION OF THE FERMENTATION BY ACETYLENE
PROCESS IN SOIL
D. H. FLATHER and E. G. BWUCHAMP+ Department of Land Resource Science, University of Guelph, Guelph, Ontario, Canada NlG 2Wl (Accepted 20 February 1992) Summary-The effect of acetylene on the fermentation of added carbon in anaerobic soil was examined. With glucose as the C source and in the absence of C2H2,the fermentation products, acetate and butyrate, were produced in nearly equimolar quantities along with copious quantities of CO, and HI. In the presence of 10 kPa of C,H,, there was significant inhibition of acetate, butyrate, CO, and H, production. T’he inhibitory effect was significantly greater on butyrate production than acetate production. In the absence of an inorganic N source, added C,H, was reduced to C2Hl, indicating the activity of the saccharolytic N,-fixing clostridia. In the presence of NH,? (nitrogenase repressed), the inhibition of fermentation due to C2Hz appeared to be alleviated. The demase in butyrate formation is suggested to result from reoxidation of NADH through the reduction of C,H2 to C,H, by nitrogenase, rather than by acetyl CoA reduction to butyrate. The potential implications of the use of C,H, in conjunction with N transformations by heterotrophic organisms, in light of these data, are discussed.
INTRODUCHON
Quantification of nitrogen fixation and denitrification rates in soil have been greatly facilitated by the advent of the acetylene reduction assay (ARA) and the acetylene blockage technique of nitrous oxide reduction to dinitrogen, respectively. The C,H, reduction and assay exploits the non-specificity of nitrogenase by reducing CIH, to C,H,. In denitrification studies, C2H, is employed to inhibit N,O reductase thus permitting an accumulation of easily and accurately quantifiable N,O. Simultaneous quantification of N fixation and denitrification is therefore possible (Yoshinari et al., 1977) as similar concentrations of C2H, are employed in both assays. Similarly, concurrent quantification of denitrification and dissimilatory nitrate reduction to ammonium (DNR) is also possible using the C,H, blockage technique (de Catanzaro et al., 1988; King and Nedwell, 1985; Oremland et al., 1984; Sorensen, 1978). Surprisingly little attention has been given to possible side effects of C2H, on denitrifying-nitrate dissimilating ecosystems when it is well documented
that C2H, is a rather unspecific inhibitor of other anaerobic microbial processes (Oremland and Capone, 1988; Payne, 1984). Kasper and Tiedje (1981) reported partial inhibition of dissimilatory nitrite reduction to NH: and N,O in the bovine rumen due to the presence of C,H,. Inhibition of DNR due to C2Hz has not been reported in soil. Growth of Clostridium pasteurianum was inhibited by 0.1 atm C2H, (Brouzes and Knowles, 1971), a concentration often employed in denitrification assays. *Author for correspondence. saa 24,9-F
Fermentative clostridia have been implicated as the primary agents of DNR in soil (Caskey and Tiedje, 1979, 1980). Intrinsic to reliable denitrification estimates, employing the C2H, blockage technique, is the assumption that C2H, does not inhibit other heterotrophs, competitive for C and NO;. The robustness of this technique was investigated by examining the effects of C2H2 on soil fermentation in three experiments. These data indicated that fermentation of added carbon was significantly inhibited in the presence of C,H,. The nature of the fermentation products measured suggested that saccharolytic, N2fixing clostridia were the organisms primarily affected. MATRR1Al.SAND METHODS
The soil used was a Conestogo silt loam collected from a continuous bromegrass plot at the Elora Research Station (20 km north of Guelph). The soil had the following characteristics: 23 g organic carbon kg-‘, 230 g sand kg-‘, 550 g silt kg-’ and 220 g clay kg-‘. At the time of sampling the soil contained 6.0 mg NO,-N kg-’ and 4.0 mg NH,,+-N kg-‘. In each of the three experiments reported here, a standard soil conditioning procedure was carried out prior to imposition of treatments. A desired quantity of frozen soil was added to 125ml Erlenmyer flasks. The flasks were capped with a serum stopper (Suba-Seal, Bamsley, England) to prevent evaporation and placed in the dark at 21°C for 5 days in order to restore conditions favorable for biological activity before treatments were imposed. 905
D. H. FLAK
906
and E. G.
Experiment 1
imposed as follows:
The aim was to determine if the presence of 10 kPa C,H, inhibits fermentation in anaerobic soil. The treatments were as follows:
(1) l.Og glucose-C kg-‘; 50mg KNO,-N kg-’ (2) same as (1) plus 10 kPa CrH, (3) 30 g ground (2 mm) wheat straw kg-‘; 50 mg KNO,-N kg-’ (4) same as (3) plus 10 kPa C2H,. The glucose and KNO, were added in a solution whereas the straw was mixed into the soil prior to addition of solution. Sufficient distilled deionized water was added to bring the soil moisture to 55Og kg-‘. Each treatment was replicated three times. Immediately following imposition of treatments, the flasks were capped and flushed with He for 20min. 10 ml of headspace gas was removed and replaced with 10ml of purified C,Hr (passed through HrSO., solution to remove acetone) (Matheson Gas Products, Whitby, Ontario) in treatments 2 and 4. The flasks were placed in the dark at 21°C and sampled at 24,48 and 96 h for CO, evolution and volatile fatty acid accumulation. Carbon dioxide in the headspace gas was analyzed by removing a 0.4 ml sample with a gas-tight syringe and injecting into a Gowmac 550 gc. equipped with a thermal conductivity detector. Following CO* analysis, volatile fatty acid concentrations were determined according to the method of Paul and Beauchamp (1989a). Experiment 2 This experiment was conducted to determine if NH: would alleviate the inhibition of fermentation observed in Experiment 1. The procedure followed was similar to that for Experiment 1 except that 30 g dry soil was weighed into each flask and Ar was used as the headspace gas. The treatments were as follows: (1) (2) (3) (4)
1.0 g same same same
BEAUCHAMP
glucose-C kg-’ as (1) plus 10 kPa CrH, as (1) plus 200 mg (NH&SO.,-N kg-’ as (3) plus 10 kPa C2H2.
(1) 30g alfalfa kg-‘; 200 mg KNOJ-N kg-‘; 10 kPa C,H, (2) 30 g alfalfa kg-‘; 10 kPa CrH, (3) 30g alfalfa kg-‘; 200mg KNO,-N kg-’ (4) 30g alfalfa kg-’ (S)-(S) identical to treatments (l)--(4) except alfalfa was replaced by 3 mg glucose-C kg-’ (9) 10 kPa C,H4 (357mg C kg-‘); 200mg KNO,-N kg-’ (10) 200mg KNO,-N kg-‘. The alfalfa consisted of tops ground to pass a 2 mm sieve. The soil in the flasks was analyzed for volatile fatty acids (VFA), NO; (for those treatments receiving 200 mg KN09-N kg-‘) and NH: with the alfalfa treatments. Following vigorous shaking of each flask for 30 s, the headspace gas was sampled for determination of C,H, concentration with an Antek 300 g.c. equipped with a flame ionization detector. Following VFA sampling, 80 ml of KC1 solution was added to the remaining soil slurry so that the mineral N extracting solution was approx. 2 M KCI. Ammonium concentration in the extract was determined by a modified method of Hanawalt and Steckel (1967). Nitrate concentration was determined following Cd reduction by Technican Method 487-77A (Technicon Industrial Systems, Tarrytown, N.Y.). RESULTS
Experiment 1 The quantities of VFA produced from 1.O g glucose kg soil-’ with or without 10 kPa of C,H,, are presented in Fig. 1. Acetate, propionate and butyrate were not detectable with either treatment after 24 h. However, after 48 h significant quantities of acetate and butyrate, but not propionate, accumulated with both treatments. With soil exposed to 10 kPa C,H,, less acetate and butyrate accumulated than soil with only He in the headspace (P < 0.05). Acetate production in C,H,-amended soil was approximately half
Without C2 Hz
Soil moisture was adjusted to 55Og H,O kg-’ by adding distilled deionized water to all flasks. In addition to COr and volatile fatty acid analyses, H, gas production was monitored. Each flask was shaken vigorously for 30 s to liberate entrapped H,. Then a 400~1 sample of the headspace gas was injected into a Hewlett-Packard 5830A gas chromatograph equipped with a thermal conductivity detector. Experiment 3 A further experiment was conducted to verify that C2H2 was being reduced to C,H, under the experimental conditions imposed. Ten treatments were
Fig. 1. Volatile fatty acid production over time in soil with glucose substrate in the presence or absence of acetylene (Experiment 1).
Fermentation inhibition by acetylene 150
1 WithCnHz
wimc* HZ -7 B
907
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WC
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Fig. 2. Carbon dioxide production over time in soil treated with glucose or straw and in the presence or absence of acetylene (Experiment 1).
that produced in the absence of C2H2. Butyrate production was strongly inhibited with CzH2 accumulating only 8 mg butyrate-C kg-’ as compared to 187 mg butyrate-C kg-’ which accumulated without C2H,. Similarly, after 96 h, butyrate accumulation was greater in soil without C,Hr, indicating that C,H, had more than a transient effect on fermentation. The pattern of CO, production from glucose metabolism reflected the differences observed in VFA accumulation (Fig. 2). When 30g wheat straw kg-’ was used as the C source, only acetate accumulated by 24 h (Fig. 3). Although the amount of acetate produced in the presence of 10 kPa C,H, was less than without C,H,, less inhibition occurred than observed with the glucose-amended soil at all subsequent sampling times. In fact, the quantities of acetate, propionate and butyrate produced were not significantly different where straw was used with or without C2H2 after
3~
VFA Time(h)
_ With CP Hz
ac pr bu 24
ecpr bu 24
acprbu 42
Time(h)
ac pr b4l 24
ac pr bu 48
ac
acprbu 72
pr bu
acprbu 40
acprbu
24
96
.wPrbu 96
Fig. 3. Volatile fatty acid production over time in soil with straw substrate in the presence or absence of acetylene (Experiment 1).
96 h. Significant differences in CO2 production were also observed only up to 48 h, and subsequently disappeared by 96 h. Experiment 2
VFA, primarily as acetate and butyrate, was produced over 72 h of incubation in soil amended with 1.Og glucose-C kg-‘, 10 kPa CIH, and 200 mg NH: N kg-’ (Fig. 4). By 48 and 72 h only the soil treated with 10 kPa C2H2 differed significantly from that with the other three treatments. In support of the findings in Experiment 1, the CzHz depressed the production
Without C2 H2 with NH.i+
Without C2 H2
With CzH2 and NH4+
ac 72 p’ bu
VFA
ec pr bu 24
ac pr bu 48
ac pr bu 72
acprbu 24
acprbu 48
acprbu 72
Fig. 4. Volatile fatty acid production over time in soil in the presence or absence of acetylene and in the presence or absence of ammonium.
D. H.
nms pew
FLATHERand E. G. BEAUCHAMP
,h)
Fig. 5. Carbon dioxide production over time in soil in the presence or absence of acetylene and in the presence or absence of ammonium.
of acetate and especially butyrate. However, in the soil amended with NH: and C2H2, acetate and butyrate production were not significantly different from that with the other two treatments without C,H, in the headspace. Similarly, CO2 production reflected the differences observed with VFA production, however soil receiving only glucose or glucose, NH: and C2H, did not accumulate significantly different quantities of CO2 in any sampling period (Fig. 5). The addition of NH: to glucose-amended soil appeared to stimulate more rapid growth, as reflected in greater VFA and CO2 production after 24 h. This enhancement was not observed in the subsequent sampling periods. The rates of H, production for each treatment are presented in Fig. 6. Hydrogen was rapidly produced in soil amended with glucose with NH: and began to be consumed after 24 h. This was indicated by the reduction in H, concentrations at 48 h (0.8 pmol g-‘) and 72 h (0.2 pmol g-l). Conversely, consumption of H, with the other three treatments began after 48 h. Hydrogen accumulation was greatest for soil amended with only glucose, whereas soils with C2H2 in the headspace produced significantly less H,. Experiment
3
Soil receiving amendments of 3 g glucose-C kg-’ and 10 kPa C2H, (treatments 5 and 6, respectively) produced significant quantities of C2H, from C,H, Table
Fig. 6. Hydrogen concentration in the headspace during time periods in the presence or absence of acetylene and in the presence or absence of ammonium.
(Table 1). The absence of C2H, production in soil amended with glucose alone (treatments 7 and 8) indicates that it was formed by the reduction of C2H, by nitrogenase. The presence of NO; in treatment 5 prevented the formation of C2H, until all the NO; had disappeared (Table 1). Ethylene rapidly accumulated by 72 h, concomitant with the disappearance of NO<. Soil amended with alfalfa as a C source did not accumulate C2HI, except for treatment 4 which transiently produced 22 nmol C2H4 G-l at 24 h (Table 1). However, in direct contrast to the analogous glucose treatments, soil amended with 30g alfalfa kg-’ and 10 kPa C,H, did not produce any C,H,. While not presented here, all soil amendments receiving alfalfa accumulated significant quantities of NH: -N by 24 h (84 mg kg-‘), 48 h (95 mg kg-‘) and 72 h (106 mg kg-‘). There was no change in concentration of C2H, added in treatment 9 (Table 1). Denitrification may have been inhibited in the presence of added CrH, (treatments 9 and 10, Table 1). DISCUSSION
The fermentation products acetate and butyrate are produced mainly by saccharolytic clostridia: Clostridium pasteurianum and C. butyricum (Jungermann et al., 1973; Crabbendam et al., 1985) which are ubiquitous in soil. These organisms effect a branched fermentation of glucose to acetate and butyrate with an energy efficiency of ATP formation
I Concentrations of ethylene and nitrate with different carbon sources and with and without acetylene or nitrate over 72 h (Experiment 3) Time (h)
Treatment no.
Carbon source
CzHz (10 kPa)
NO, (200mg N kg-‘)
I 2 3 4 5 6 7 8 9 IO
alfalfa alfalfa alfalfa alfalfa glucose glucose glucose glucose GH, none
+ + + + -
+ +
-
+ -I+ +
24 48 72 (nmol C,H, g-‘) 0 0 0 22 0 63 O 0 91,051 0
0 0 0 0 0 610 0 0 10,500 0
0 0 0 0 144 1214 0 0 10,550 0
$00,.
::
20 0 19 0 130 0 120 0 146 144
0 0 0 0 52 0 45 0 144 140
N kg”) 0 0 0 0 0 0 0 0 135 113
Fermentation inhibition by acetylene of 62% (Thauer et al., 1977; Crabbendam et al., 1985). If only acetate is produced, the energy efficiency increases to 85%. This is apparently incompatible with the entropy requirements of clostridal metabolism; hence, acetate is never the sole fermentation product produced (Thauer et al., 1977). Acetate: butyrate molar ratios from glucose metabolism by pure cultures of C. pasteurianum or C. butyricum have been reported to range from 0.5 to 1.0 (Daesch and Mortenson, 1967; Jungermann, et al., 1973; Crabbendam et al., 1985). Acetate: butyrate molar ratios observed in the above experiments in soil ranged from 1.2 to 1.5 indicating that saccharolytic clostridia were the organisms predominantly responsible for the production of VFA under the experimental conditions employed. With glucose as the C source, the presence of 10 kPa C,H, significantly reduced acetate and butyrate production in anaerobic soil as compared to soil without C,H,. Moreover, butyrate production was more strongly inhibited than acetate production, such that acetate was the primary fermentation product in C,H,-amended soil. Conversely, Watanabe and de Guzman (1980) reported that more acetate accumulated in C,H,-amended soil than without C2H2 as a result of the oxidation of C2Hz forming acetate. In our study, the effect of C2H, was one of inhibition not stimulation. Employing the C2H, blockage of N,O reduction to N,, Paul and Beauchamp (1989b) found acetate, propionate and butyrate to be oxidizable C sources for denitrifying bacteria. Paul and Beauchamp (1989b) also observed that acetate was the dominant VFA produced in anaerobic Camended soil. However, in light of the above, it appears that this latter finding may have been an artifact of the experimental technique employing C2H,. When straw was used as the C source, mainly acetate accumulated in agreement with the results of Lynch and Gun (1978). The wheat straw used in their study was high in cellulose and lignin (de Catanzaro et al., 1987). Once the readily metabolizable portion of the C in straw is consumed, continued decomposition requires the action of cellulase. Rice and Paul (1971) observed that, under strictly anaerobic conditions with straw as substrate, N,-fixation rates were extremely low. This indicated that the cellulose and hemicellulose in the straw could not be utilized by the N,-fixing clostridia (Rice and Paul, 1971). Anaerobes, isolated in their study fermented hemicellulose very slowly and they concluded that aerobic organisms with cellulolytic activity were responsible for providing the clostridia with the products of cellulose activity: cellobiose and glucose. The observed difference in inhibitory action of C+H, and in the fermentation products with glucose and straw indicates that the dominant communities of organisms varied with the C source. The influence of the saccharolytic clostridia on the metabolism of added C in soil was greater with glucose than with straw as carbon source.
909
Carbon dioxide evolution from glucose-amended soil was also inhibited in the presence of C,H,. Brouzes and Knowles (1971) reported that C2H2 (approx. 10 kPa) inhibited cell division of C. pasteurianum in pure culture and repressed CO2 production associated with microbial growth in a glucoseamended, anaerobic sandy loam soil. Growth and CO2 production resumed after complete reduction of C,H, to C2H,. However, nitrogenase-repressed cells, grown on media containing 339 pg NH:-N ml-‘, were unable to overcome the inhibitory effect even after all the C,H, had been removed (Brouzes and Knowles, 1971). The discrepancy between these re.sults and those reported here may reflect the difficulty often encountered when extrapolating results from pure culture to low-activity materials such as soils (Knowles and Denike, 1974). Acetate, butyrate and CO, production were not suppressed in the presence of C,H, when NH.,+ was present, in direct contrast to the significant suppression of acetate production, and in particular, butyrate production in glucose-amended soil with acetylene. The explanation for this suppression is centered around the activity of nitrogenase reducing C2H2 to C*H,. Reduced ferredoxin and NADH are the physiological electron donors for nitrogenase in N,-fixing clostridia (Jungermann et al., 1974). Jungermann et al. (1974) reported that up to 25% of the electrons used for N, reduction can be donated directly by NADH. The NADH produced during glucose oxidation is reoxidized by the reduction of
A
NADH--J
Ferredoin +
NADPH
Amtete
Fig. 7. Proposed electron pathways during the metabolism of glucose in the absence (A) and presence (B) of acetylene (modified from Jungermann er al., 1973).
910
D. H. FLATHERand E. G. BEAUCHAMP
ferredoxin, catalyzed by NADH-ferredoxin oxidoreductase or by acetyl CoA reduction via butyryl CoA to butyrate (Jungermann et al., 1974). The result of either process is the regeneration of NAD + which can be reused in glycolysis [Fig. 7(A)]. However, when C2H, is present, stimulating the synthesis and activity of nitrogenase, the NADH that is produced during dehydrogenation of glyceraldehyde phosphate, is reoxidized mainly during the reduction of ferredoxin and through donation of electrons directly to nitrogenase. Thus a balanced oxidation-reduction is achieved without the formation of butyrate. Figure 7(B) schematically illustrates the proposed manner in which butyrate production is repressed in the presence of C2H2. Repression of nitrogenase activity should therefore result in quantities of butyrate produced similar to those observed without C2Hz in the headspace. In support of this hypothesis, acetate and butyrate production were not significantly different in treatments receiving glucose alone, glucoseNH: and glucoseNH,+-C,H,. However, butyrate production was greatly inhibited in glucose-C,H,-amended soil. Work indirectly supporting the above hypothesis has been with external electron acceptors that reoxidase NADH preferentially over NADH reoxidation through butyrate production. Ishimota et al. (1974), Hasan and Hall (1975) and Keith et al. (1982) observed a shift in fermentation of Clostridium spp when nitrate was present as an external electron acceptor. Nitrate serves as a reoxidant of NADH during NO; reduction to NH: resulting in a similar shift in fermentation products away from butyrate production to acetate. Morris and O’Brien (1971) observed that 0, inhibited growth of C. acetobutyficum mainly by acting as an external electron acceptor for NADH reoxidation. Butyrate production ceased in the presence of 0, and growth stopped, indicating that NADH and ferredoxin were being reoxidized by 0,. It was construed that the loss of “reducing power”, as evidenced by restricted production of butyrate, occurs concomitant to the inhibition of growth (Morris and O’Brien, 1971). This idea could be extrapolated to the data presented here to suggest that CrH, reduction to C2H4 drains the N,-fixing clostridia of reducing power, both as NADH and NADPH; hence, the restriction in growth as evidenced by reduced CO* production. Hydrogen production in C,H,-amended soil was significantly inhibited compared to soil without C,H2, regardless of NH: addition. Although NH: appeared to alleviate the effect of CzH2 on CO2 and VFA production, hydrogen production remained inhibited. This finding is not in disagreement with the proposed hypothesis, as C2H, has a separate inhibitory effect upon hydrogenase. Hydrogenase catalyzes the reduction of protons to Hz, coupled to the reoxidation of reduced ferredoxin, thus maintaining a constant level of oxidized ferredoxin within the cell (Smith et al., 1976). Acetylene has been shown to
inhibit this ATP-independent hydrogenase in Klebsiella pneumoniae (Smith et al., 1976). What is not clear from the above experiments is whether or not C2Hz is inhibiting hydrogenase, or whether ferredoxin reoxidation via nitrogenase is predominant over ferredoxin-reoxidation via hydrogenase? Hydrogenase activity has been shown to be greatly reduced in C. butyricum when nitrate is present as external electron acceptor (Keith et al., 1982). As previously mentioned, NO; serves as reoxidant of NADH resulting in a shift in fermentation products away from butyrate with a concurrent reduction in H, production (Ishimota et al., 1974; Hasan and Hall, 1975; Keith et al., 1982). Therefore, NADH reoxidation via C2H2 reduction may explain a portion of the observed inhibition of H, production. It has been demonstrated that the addition of CrH, in concentrations similar to that employed in the C,H, blockage of N,O reduction in denitrification studies significantly inhibits the fermentation of metabolizable C in soil under anaerobic conditions. When an inorganic N source was unavailable, significant quantities of C2H, accumulated, indicating that N,-fixation was an active process in this soil under experimental conditions. Similarly, the production of nearly equimolar quantities of acetate and butyrate, along with production of copious quantities of H2 and CO2 suggested a high activity of saccharolytic nitrogen-fixing Clostridium spp under anaerobic conditions and the absence of a terminal oxidant. An important implication of this research is that restriction of growth by C2H, reduces total C consumption by the anaerobic bacteria. If for a rough calculation, the percentage of C converted to cellular material in anaerobic bacteria is assumed to be approx. 10% (Daesch and Mortenson, 1967), then the total amount of C utilized in the presence of CrH, was approx. 50% less than without CzH2. This would result in substantially more C being available to other competitive heterotrophs when C,Hr is present. This has considerable bearing on the continued use of CrH, in studies of the partitioning of NO< between denitrification and dissimilatory NO; reduction to NH: as the fermentative bacteria, and in particular the clostridia, have been shown to be primarily responsible for NO; reduction to NH,++ in soil. As such, the use of CrHr may overestimate the denitrification potential of C rich environments. Acknowledgements-Funding by the Natural Sciences and Engineering Research Council of Canada and assistance of S. Dorland are gratefully acknowledged.
REFERENCES Brouzes R. and Knowles R. (1971) Inhibition of growth of Clostridium pasteurianum by acetylene, implication for nitrogen fixation assay. Canadian Journal of Microbiology 17, 1483-1489. Caskey W. H. and Tiedje J. M. (1979) Evidence for clostridia as agents of dissimilatory reduction of nitrate
Fermentation inhiblition by acetylene to ammonium in soils. Soil Science Society of America Journal 43, 93 l-936.
Caskey W. H. and Tiedje J. M. (1980) The reduction of nitrate to ammonium by a Closrtidium sp. isolated from soil. Journal of General Microbiology 199, 217-223. de Catanzaro J. B., Beauchamp E. G. and Drury C. F. (1987) Denitrification versus dissimilatory nitrate reduction in soil with alfalfa, straw, glucose and sulfide treatments. Soil Biology & Biochemistry 19, 583-587. Crabbendam P. M., Neijssel 0. M. and Tempest D. W. (1985) Metabolic and energetic aspects of the growth of Closttidium butyticum on glucose in chemostat culture. Archives of Microbiology 142, 375-382.
Daesch G. and Mortenson L. E. (1967) Sucrose catabolism in Closttidium pasreutianum and its relation to N, fixation. Journal of Bacretiology 96, 34&351. Hanawalt R. B. and Steckel J. E. (1967) Ammonium determination by automated distillation from soil suspensions and colored solutions. In Aulomation in Analyrical Chemistry, Technicon Symposium I, pp. 133-I 36. Medied Inc., White Plains, N.Y. Hasan S. M. and Hall J. B. (1975) The physiological function of nitrate reduction in Closttidium petftingens. Journal of General Microbiology 87, 12&128.
Ishimota M., Umeyama M. and Chiba S. (1974) Alteration of fermentation products from butyrate to acetate by nitrate reduction in Closttidiumpetftingens. Zeitschtiftfit Allgemeine Miktobiologie 14, 115-12 1. Jungermann K., Kirchniawy H., Katz N. and Thauer R. K. (1974) NADH, a physiological electron’ donor in clostridial nitrogen fixation. FEBS L&lets 43, 203-206. Jungermann K., -Thauer R. K., Leimonstoil G. and Decker K. (1973) Function of reduced uvridine nucleotide-feriedoxin oxidoreductases in sac&rolytic clostridia. Biochimica and Biophysics Acra 305, 268-280. Kaspar H. F. and Tiedji J. M. (1981) Dissimilatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and the effect of acetylene. Applied and Environmental Microbiology 41, 705-709.
Keith S. M., MacFarlane G. T. and Herbert R. A. (1982) Dissimilatory nitrate reduction by a strain of Closttidium butyticum isolated from estuarine sediments. Archives of Microbiology 132, 62-66.
King D. and Nedwell D. B. (1985) The influence of nitrate concentration upon the end-products of nitrate dissimilation by bacteria in anaerobic salt marsh sediment. FEMS Microbiological Ecology 31, 23-28.
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Knowles R. and Denike D. (1974) Effects of ammonium-, nitrite-, and nitrate-nitrogen on anaerobic nitrogenase activity in soil. Soil Biology & Biochemistry 6, 353-358. Lynch J. M. and Gunn K. B. (1978) Use of a chemostat to study decomposition of wheat straw in soil slurries. Journal of Soil Science 29, 551-556.
Morris J. G. and O’Brien R. W. (1971) Oxygen and clostridia: a review. In Spore Research 1971 (A. N. Barker, G. W. Gould and J. Wolf, Eds). Academic Press, London. Oremland R. S. and Capone D. G. (1988) Use of “specific” inhibitors in biogeochemistry and microbial ecology. In Advances in Microbiological Ecology IO (M. Alexander, Ed.), p. 285. Plenum Press, New York. Oremland R. S., Umberger C., Cubbertson C. W. and Smith R. L. (1984) Denitrification in San Francisco Bay intertidal sediments. Applied and Envitonmenral Microbiology 47, 1106-1112.
Paul J. W. and Beauchamp E. G. (1989a) Rapid extraction and analysis of volatile fatty acids in soil. Communicarions in Soil Science and Planr Analysis 20, 85-94. Paul J. W. and Beauchamp E. G. (1989b) Denitrification and fermentation in plait residueiamended soil. Biology and Fertility of Soils 7, 303-309.
Payne W. J. (1984) Influence of acetylene on microbial and enzymatic assays. Journal of Microbiological Methods 2, 117-133.
Rice W. A. and Paul E. A. (1971) The organisms and biological processes involved in asymbiotic nitrogen fixation in waterlogged soil amended with straw. Canadian Journal of Microbiology 18, 715-723. Smith L. A., Hill S. and Yates M. G. (1976) Inhibition by acetylene of conventional hydrogenase in nitrogen-fixing bacteria. Nature 262, 209-210. Sorensen J. (1978) Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Applied and Environmental Microbiology 35, 301-305.
Thauer R. K., Jungermann K. and Decker K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriological Reviews 41, 100-180.
Watanabe I. and de Guzman M. R. (1980) Effect of nitrate on acetylene disappearance from anaerobic soil. Soil Biology & Biochemisrty 12, 193-194.
Yoshinari T., Hynes R. and Knowles R. (1977) Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biology & Biochemistry 9, 177-183.
003&0717/92 SS.00 + 0.00 Copyright 0 1992 Pergamon Press Ltd
INHIBITION OF THE FERMENTATION BY ACETYLENE
PROCESS IN SOIL
D. H. FLATHER and E. G. BWUCHAMP+ Department of Land Resource Science, University of Guelph, Guelph, Ontario, Canada NlG 2Wl (Accepted 20 February 1992) Summary-The effect of acetylene on the fermentation of added carbon in anaerobic soil was examined. With glucose as the C source and in the absence of C2H2,the fermentation products, acetate and butyrate, were produced in nearly equimolar quantities along with copious quantities of CO, and HI. In the presence of 10 kPa of C,H,, there was significant inhibition of acetate, butyrate, CO, and H, production. T’he inhibitory effect was significantly greater on butyrate production than acetate production. In the absence of an inorganic N source, added C,H, was reduced to C2Hl, indicating the activity of the saccharolytic N,-fixing clostridia. In the presence of NH,? (nitrogenase repressed), the inhibition of fermentation due to C2Hz appeared to be alleviated. The demase in butyrate formation is suggested to result from reoxidation of NADH through the reduction of C,H2 to C,H, by nitrogenase, rather than by acetyl CoA reduction to butyrate. The potential implications of the use of C,H, in conjunction with N transformations by heterotrophic organisms, in light of these data, are discussed.
INTRODUCHON
Quantification of nitrogen fixation and denitrification rates in soil have been greatly facilitated by the advent of the acetylene reduction assay (ARA) and the acetylene blockage technique of nitrous oxide reduction to dinitrogen, respectively. The C,H, reduction and assay exploits the non-specificity of nitrogenase by reducing CIH, to C,H,. In denitrification studies, C2H, is employed to inhibit N,O reductase thus permitting an accumulation of easily and accurately quantifiable N,O. Simultaneous quantification of N fixation and denitrification is therefore possible (Yoshinari et al., 1977) as similar concentrations of C2H, are employed in both assays. Similarly, concurrent quantification of denitrification and dissimilatory nitrate reduction to ammonium (DNR) is also possible using the C,H, blockage technique (de Catanzaro et al., 1988; King and Nedwell, 1985; Oremland et al., 1984; Sorensen, 1978). Surprisingly little attention has been given to possible side effects of C2H, on denitrifying-nitrate dissimilating ecosystems when it is well documented
that C2H, is a rather unspecific inhibitor of other anaerobic microbial processes (Oremland and Capone, 1988; Payne, 1984). Kasper and Tiedje (1981) reported partial inhibition of dissimilatory nitrite reduction to NH: and N,O in the bovine rumen due to the presence of C,H,. Inhibition of DNR due to C2Hz has not been reported in soil. Growth of Clostridium pasteurianum was inhibited by 0.1 atm C2H, (Brouzes and Knowles, 1971), a concentration often employed in denitrification assays. *Author for correspondence. saa 24,9-F
Fermentative clostridia have been implicated as the primary agents of DNR in soil (Caskey and Tiedje, 1979, 1980). Intrinsic to reliable denitrification estimates, employing the C2H, blockage technique, is the assumption that C2H, does not inhibit other heterotrophs, competitive for C and NO;. The robustness of this technique was investigated by examining the effects of C2H2 on soil fermentation in three experiments. These data indicated that fermentation of added carbon was significantly inhibited in the presence of C,H,. The nature of the fermentation products measured suggested that saccharolytic, N2fixing clostridia were the organisms primarily affected. MATRR1Al.SAND METHODS
The soil used was a Conestogo silt loam collected from a continuous bromegrass plot at the Elora Research Station (20 km north of Guelph). The soil had the following characteristics: 23 g organic carbon kg-‘, 230 g sand kg-‘, 550 g silt kg-’ and 220 g clay kg-‘. At the time of sampling the soil contained 6.0 mg NO,-N kg-’ and 4.0 mg NH,,+-N kg-‘. In each of the three experiments reported here, a standard soil conditioning procedure was carried out prior to imposition of treatments. A desired quantity of frozen soil was added to 125ml Erlenmyer flasks. The flasks were capped with a serum stopper (Suba-Seal, Bamsley, England) to prevent evaporation and placed in the dark at 21°C for 5 days in order to restore conditions favorable for biological activity before treatments were imposed. 905
D. H. FLAK
906
and E. G.
Experiment 1
imposed as follows:
The aim was to determine if the presence of 10 kPa C,H, inhibits fermentation in anaerobic soil. The treatments were as follows:
(1) l.Og glucose-C kg-‘; 50mg KNO,-N kg-’ (2) same as (1) plus 10 kPa CrH, (3) 30 g ground (2 mm) wheat straw kg-‘; 50 mg KNO,-N kg-’ (4) same as (3) plus 10 kPa C2H,. The glucose and KNO, were added in a solution whereas the straw was mixed into the soil prior to addition of solution. Sufficient distilled deionized water was added to bring the soil moisture to 55Og kg-‘. Each treatment was replicated three times. Immediately following imposition of treatments, the flasks were capped and flushed with He for 20min. 10 ml of headspace gas was removed and replaced with 10ml of purified C,Hr (passed through HrSO., solution to remove acetone) (Matheson Gas Products, Whitby, Ontario) in treatments 2 and 4. The flasks were placed in the dark at 21°C and sampled at 24,48 and 96 h for CO, evolution and volatile fatty acid accumulation. Carbon dioxide in the headspace gas was analyzed by removing a 0.4 ml sample with a gas-tight syringe and injecting into a Gowmac 550 gc. equipped with a thermal conductivity detector. Following CO* analysis, volatile fatty acid concentrations were determined according to the method of Paul and Beauchamp (1989a). Experiment 2 This experiment was conducted to determine if NH: would alleviate the inhibition of fermentation observed in Experiment 1. The procedure followed was similar to that for Experiment 1 except that 30 g dry soil was weighed into each flask and Ar was used as the headspace gas. The treatments were as follows: (1) (2) (3) (4)
1.0 g same same same
BEAUCHAMP
glucose-C kg-’ as (1) plus 10 kPa CrH, as (1) plus 200 mg (NH&SO.,-N kg-’ as (3) plus 10 kPa C2H2.
(1) 30g alfalfa kg-‘; 200 mg KNOJ-N kg-‘; 10 kPa C,H, (2) 30 g alfalfa kg-‘; 10 kPa CrH, (3) 30g alfalfa kg-‘; 200mg KNO,-N kg-’ (4) 30g alfalfa kg-’ (S)-(S) identical to treatments (l)--(4) except alfalfa was replaced by 3 mg glucose-C kg-’ (9) 10 kPa C,H4 (357mg C kg-‘); 200mg KNO,-N kg-’ (10) 200mg KNO,-N kg-‘. The alfalfa consisted of tops ground to pass a 2 mm sieve. The soil in the flasks was analyzed for volatile fatty acids (VFA), NO; (for those treatments receiving 200 mg KN09-N kg-‘) and NH: with the alfalfa treatments. Following vigorous shaking of each flask for 30 s, the headspace gas was sampled for determination of C,H, concentration with an Antek 300 g.c. equipped with a flame ionization detector. Following VFA sampling, 80 ml of KC1 solution was added to the remaining soil slurry so that the mineral N extracting solution was approx. 2 M KCI. Ammonium concentration in the extract was determined by a modified method of Hanawalt and Steckel (1967). Nitrate concentration was determined following Cd reduction by Technican Method 487-77A (Technicon Industrial Systems, Tarrytown, N.Y.). RESULTS
Experiment 1 The quantities of VFA produced from 1.O g glucose kg soil-’ with or without 10 kPa of C,H,, are presented in Fig. 1. Acetate, propionate and butyrate were not detectable with either treatment after 24 h. However, after 48 h significant quantities of acetate and butyrate, but not propionate, accumulated with both treatments. With soil exposed to 10 kPa C,H,, less acetate and butyrate accumulated than soil with only He in the headspace (P < 0.05). Acetate production in C,H,-amended soil was approximately half
Without C2 Hz
Soil moisture was adjusted to 55Og H,O kg-’ by adding distilled deionized water to all flasks. In addition to COr and volatile fatty acid analyses, H, gas production was monitored. Each flask was shaken vigorously for 30 s to liberate entrapped H,. Then a 400~1 sample of the headspace gas was injected into a Hewlett-Packard 5830A gas chromatograph equipped with a thermal conductivity detector. Experiment 3 A further experiment was conducted to verify that C2H2 was being reduced to C,H, under the experimental conditions imposed. Ten treatments were
Fig. 1. Volatile fatty acid production over time in soil with glucose substrate in the presence or absence of acetylene (Experiment 1).
Fermentation inhibition by acetylene 150
1 WithCnHz
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907
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Fig. 2. Carbon dioxide production over time in soil treated with glucose or straw and in the presence or absence of acetylene (Experiment 1).
that produced in the absence of C2H2. Butyrate production was strongly inhibited with CzH2 accumulating only 8 mg butyrate-C kg-’ as compared to 187 mg butyrate-C kg-’ which accumulated without C2H,. Similarly, after 96 h, butyrate accumulation was greater in soil without C,Hr, indicating that C,H, had more than a transient effect on fermentation. The pattern of CO, production from glucose metabolism reflected the differences observed in VFA accumulation (Fig. 2). When 30g wheat straw kg-’ was used as the C source, only acetate accumulated by 24 h (Fig. 3). Although the amount of acetate produced in the presence of 10 kPa C,H, was less than without C,H,, less inhibition occurred than observed with the glucose-amended soil at all subsequent sampling times. In fact, the quantities of acetate, propionate and butyrate produced were not significantly different where straw was used with or without C2H2 after
3~
VFA Time(h)
_ With CP Hz
ac pr bu 24
ecpr bu 24
acprbu 42
Time(h)
ac pr b4l 24
ac pr bu 48
ac
acprbu 72
pr bu
acprbu 40
acprbu
24
96
.wPrbu 96
Fig. 3. Volatile fatty acid production over time in soil with straw substrate in the presence or absence of acetylene (Experiment 1).
96 h. Significant differences in CO2 production were also observed only up to 48 h, and subsequently disappeared by 96 h. Experiment 2
VFA, primarily as acetate and butyrate, was produced over 72 h of incubation in soil amended with 1.Og glucose-C kg-‘, 10 kPa CIH, and 200 mg NH: N kg-’ (Fig. 4). By 48 and 72 h only the soil treated with 10 kPa C2H2 differed significantly from that with the other three treatments. In support of the findings in Experiment 1, the CzHz depressed the production
Without C2 H2 with NH.i+
Without C2 H2
With CzH2 and NH4+
ac 72 p’ bu
VFA
ec pr bu 24
ac pr bu 48
ac pr bu 72
acprbu 24
acprbu 48
acprbu 72
Fig. 4. Volatile fatty acid production over time in soil in the presence or absence of acetylene and in the presence or absence of ammonium.
D. H.
nms pew
FLATHERand E. G. BEAUCHAMP
,h)
Fig. 5. Carbon dioxide production over time in soil in the presence or absence of acetylene and in the presence or absence of ammonium.
of acetate and especially butyrate. However, in the soil amended with NH: and C2H2, acetate and butyrate production were not significantly different from that with the other two treatments without C,H, in the headspace. Similarly, CO2 production reflected the differences observed with VFA production, however soil receiving only glucose or glucose, NH: and C2H, did not accumulate significantly different quantities of CO2 in any sampling period (Fig. 5). The addition of NH: to glucose-amended soil appeared to stimulate more rapid growth, as reflected in greater VFA and CO2 production after 24 h. This enhancement was not observed in the subsequent sampling periods. The rates of H, production for each treatment are presented in Fig. 6. Hydrogen was rapidly produced in soil amended with glucose with NH: and began to be consumed after 24 h. This was indicated by the reduction in H, concentrations at 48 h (0.8 pmol g-‘) and 72 h (0.2 pmol g-l). Conversely, consumption of H, with the other three treatments began after 48 h. Hydrogen accumulation was greatest for soil amended with only glucose, whereas soils with C2H2 in the headspace produced significantly less H,. Experiment
3
Soil receiving amendments of 3 g glucose-C kg-’ and 10 kPa C2H, (treatments 5 and 6, respectively) produced significant quantities of C2H, from C,H, Table
Fig. 6. Hydrogen concentration in the headspace during time periods in the presence or absence of acetylene and in the presence or absence of ammonium.
(Table 1). The absence of C2H, production in soil amended with glucose alone (treatments 7 and 8) indicates that it was formed by the reduction of C2H, by nitrogenase. The presence of NO; in treatment 5 prevented the formation of C2H, until all the NO; had disappeared (Table 1). Ethylene rapidly accumulated by 72 h, concomitant with the disappearance of NO<. Soil amended with alfalfa as a C source did not accumulate C2HI, except for treatment 4 which transiently produced 22 nmol C2H4 G-l at 24 h (Table 1). However, in direct contrast to the analogous glucose treatments, soil amended with 30g alfalfa kg-’ and 10 kPa C,H, did not produce any C,H,. While not presented here, all soil amendments receiving alfalfa accumulated significant quantities of NH: -N by 24 h (84 mg kg-‘), 48 h (95 mg kg-‘) and 72 h (106 mg kg-‘). There was no change in concentration of C2H, added in treatment 9 (Table 1). Denitrification may have been inhibited in the presence of added CrH, (treatments 9 and 10, Table 1). DISCUSSION
The fermentation products acetate and butyrate are produced mainly by saccharolytic clostridia: Clostridium pasteurianum and C. butyricum (Jungermann et al., 1973; Crabbendam et al., 1985) which are ubiquitous in soil. These organisms effect a branched fermentation of glucose to acetate and butyrate with an energy efficiency of ATP formation
I Concentrations of ethylene and nitrate with different carbon sources and with and without acetylene or nitrate over 72 h (Experiment 3) Time (h)
Treatment no.
Carbon source
CzHz (10 kPa)
NO, (200mg N kg-‘)
I 2 3 4 5 6 7 8 9 IO
alfalfa alfalfa alfalfa alfalfa glucose glucose glucose glucose GH, none
+ + + + -
+ +
-
+ -I+ +
24 48 72 (nmol C,H, g-‘) 0 0 0 22 0 63 O 0 91,051 0
0 0 0 0 0 610 0 0 10,500 0
0 0 0 0 144 1214 0 0 10,550 0
$00,.
::
20 0 19 0 130 0 120 0 146 144
0 0 0 0 52 0 45 0 144 140
N kg”) 0 0 0 0 0 0 0 0 135 113
Fermentation inhibition by acetylene of 62% (Thauer et al., 1977; Crabbendam et al., 1985). If only acetate is produced, the energy efficiency increases to 85%. This is apparently incompatible with the entropy requirements of clostridal metabolism; hence, acetate is never the sole fermentation product produced (Thauer et al., 1977). Acetate: butyrate molar ratios from glucose metabolism by pure cultures of C. pasteurianum or C. butyricum have been reported to range from 0.5 to 1.0 (Daesch and Mortenson, 1967; Jungermann, et al., 1973; Crabbendam et al., 1985). Acetate: butyrate molar ratios observed in the above experiments in soil ranged from 1.2 to 1.5 indicating that saccharolytic clostridia were the organisms predominantly responsible for the production of VFA under the experimental conditions employed. With glucose as the C source, the presence of 10 kPa C,H, significantly reduced acetate and butyrate production in anaerobic soil as compared to soil without C,H,. Moreover, butyrate production was more strongly inhibited than acetate production, such that acetate was the primary fermentation product in C,H,-amended soil. Conversely, Watanabe and de Guzman (1980) reported that more acetate accumulated in C,H,-amended soil than without C2H2 as a result of the oxidation of C2Hz forming acetate. In our study, the effect of C2H, was one of inhibition not stimulation. Employing the C2H, blockage of N,O reduction to N,, Paul and Beauchamp (1989b) found acetate, propionate and butyrate to be oxidizable C sources for denitrifying bacteria. Paul and Beauchamp (1989b) also observed that acetate was the dominant VFA produced in anaerobic Camended soil. However, in light of the above, it appears that this latter finding may have been an artifact of the experimental technique employing C2H,. When straw was used as the C source, mainly acetate accumulated in agreement with the results of Lynch and Gun (1978). The wheat straw used in their study was high in cellulose and lignin (de Catanzaro et al., 1987). Once the readily metabolizable portion of the C in straw is consumed, continued decomposition requires the action of cellulase. Rice and Paul (1971) observed that, under strictly anaerobic conditions with straw as substrate, N,-fixation rates were extremely low. This indicated that the cellulose and hemicellulose in the straw could not be utilized by the N,-fixing clostridia (Rice and Paul, 1971). Anaerobes, isolated in their study fermented hemicellulose very slowly and they concluded that aerobic organisms with cellulolytic activity were responsible for providing the clostridia with the products of cellulose activity: cellobiose and glucose. The observed difference in inhibitory action of C+H, and in the fermentation products with glucose and straw indicates that the dominant communities of organisms varied with the C source. The influence of the saccharolytic clostridia on the metabolism of added C in soil was greater with glucose than with straw as carbon source.
909
Carbon dioxide evolution from glucose-amended soil was also inhibited in the presence of C,H,. Brouzes and Knowles (1971) reported that C2H2 (approx. 10 kPa) inhibited cell division of C. pasteurianum in pure culture and repressed CO2 production associated with microbial growth in a glucoseamended, anaerobic sandy loam soil. Growth and CO2 production resumed after complete reduction of C,H, to C2H,. However, nitrogenase-repressed cells, grown on media containing 339 pg NH:-N ml-‘, were unable to overcome the inhibitory effect even after all the C,H, had been removed (Brouzes and Knowles, 1971). The discrepancy between these re.sults and those reported here may reflect the difficulty often encountered when extrapolating results from pure culture to low-activity materials such as soils (Knowles and Denike, 1974). Acetate, butyrate and CO, production were not suppressed in the presence of C,H, when NH.,+ was present, in direct contrast to the significant suppression of acetate production, and in particular, butyrate production in glucose-amended soil with acetylene. The explanation for this suppression is centered around the activity of nitrogenase reducing C2H2 to C*H,. Reduced ferredoxin and NADH are the physiological electron donors for nitrogenase in N,-fixing clostridia (Jungermann et al., 1974). Jungermann et al. (1974) reported that up to 25% of the electrons used for N, reduction can be donated directly by NADH. The NADH produced during glucose oxidation is reoxidized by the reduction of
A
NADH--J
Ferredoin +
NADPH
Amtete
Fig. 7. Proposed electron pathways during the metabolism of glucose in the absence (A) and presence (B) of acetylene (modified from Jungermann er al., 1973).
910
D. H. FLATHERand E. G. BEAUCHAMP
ferredoxin, catalyzed by NADH-ferredoxin oxidoreductase or by acetyl CoA reduction via butyryl CoA to butyrate (Jungermann et al., 1974). The result of either process is the regeneration of NAD + which can be reused in glycolysis [Fig. 7(A)]. However, when C2H, is present, stimulating the synthesis and activity of nitrogenase, the NADH that is produced during dehydrogenation of glyceraldehyde phosphate, is reoxidized mainly during the reduction of ferredoxin and through donation of electrons directly to nitrogenase. Thus a balanced oxidation-reduction is achieved without the formation of butyrate. Figure 7(B) schematically illustrates the proposed manner in which butyrate production is repressed in the presence of C2H2. Repression of nitrogenase activity should therefore result in quantities of butyrate produced similar to those observed without C2Hz in the headspace. In support of this hypothesis, acetate and butyrate production were not significantly different in treatments receiving glucose alone, glucoseNH: and glucoseNH,+-C,H,. However, butyrate production was greatly inhibited in glucose-C,H,-amended soil. Work indirectly supporting the above hypothesis has been with external electron acceptors that reoxidase NADH preferentially over NADH reoxidation through butyrate production. Ishimota et al. (1974), Hasan and Hall (1975) and Keith et al. (1982) observed a shift in fermentation of Clostridium spp when nitrate was present as an external electron acceptor. Nitrate serves as a reoxidant of NADH during NO; reduction to NH: resulting in a similar shift in fermentation products away from butyrate production to acetate. Morris and O’Brien (1971) observed that 0, inhibited growth of C. acetobutyficum mainly by acting as an external electron acceptor for NADH reoxidation. Butyrate production ceased in the presence of 0, and growth stopped, indicating that NADH and ferredoxin were being reoxidized by 0,. It was construed that the loss of “reducing power”, as evidenced by restricted production of butyrate, occurs concomitant to the inhibition of growth (Morris and O’Brien, 1971). This idea could be extrapolated to the data presented here to suggest that CrH, reduction to C2H4 drains the N,-fixing clostridia of reducing power, both as NADH and NADPH; hence, the restriction in growth as evidenced by reduced CO* production. Hydrogen production in C,H,-amended soil was significantly inhibited compared to soil without C,H2, regardless of NH: addition. Although NH: appeared to alleviate the effect of CzH2 on CO2 and VFA production, hydrogen production remained inhibited. This finding is not in disagreement with the proposed hypothesis, as C2H, has a separate inhibitory effect upon hydrogenase. Hydrogenase catalyzes the reduction of protons to Hz, coupled to the reoxidation of reduced ferredoxin, thus maintaining a constant level of oxidized ferredoxin within the cell (Smith et al., 1976). Acetylene has been shown to
inhibit this ATP-independent hydrogenase in Klebsiella pneumoniae (Smith et al., 1976). What is not clear from the above experiments is whether or not C2Hz is inhibiting hydrogenase, or whether ferredoxin reoxidation via nitrogenase is predominant over ferredoxin-reoxidation via hydrogenase? Hydrogenase activity has been shown to be greatly reduced in C. butyricum when nitrate is present as external electron acceptor (Keith et al., 1982). As previously mentioned, NO; serves as reoxidant of NADH resulting in a shift in fermentation products away from butyrate with a concurrent reduction in H, production (Ishimota et al., 1974; Hasan and Hall, 1975; Keith et al., 1982). Therefore, NADH reoxidation via C2H2 reduction may explain a portion of the observed inhibition of H, production. It has been demonstrated that the addition of CrH, in concentrations similar to that employed in the C,H, blockage of N,O reduction in denitrification studies significantly inhibits the fermentation of metabolizable C in soil under anaerobic conditions. When an inorganic N source was unavailable, significant quantities of C2H, accumulated, indicating that N,-fixation was an active process in this soil under experimental conditions. Similarly, the production of nearly equimolar quantities of acetate and butyrate, along with production of copious quantities of H2 and CO2 suggested a high activity of saccharolytic nitrogen-fixing Clostridium spp under anaerobic conditions and the absence of a terminal oxidant. An important implication of this research is that restriction of growth by C2H, reduces total C consumption by the anaerobic bacteria. If for a rough calculation, the percentage of C converted to cellular material in anaerobic bacteria is assumed to be approx. 10% (Daesch and Mortenson, 1967), then the total amount of C utilized in the presence of CrH, was approx. 50% less than without CzH2. This would result in substantially more C being available to other competitive heterotrophs when C,Hr is present. This has considerable bearing on the continued use of CrH, in studies of the partitioning of NO< between denitrification and dissimilatory NO; reduction to NH: as the fermentative bacteria, and in particular the clostridia, have been shown to be primarily responsible for NO; reduction to NH,++ in soil. As such, the use of CrHr may overestimate the denitrification potential of C rich environments. Acknowledgements-Funding by the Natural Sciences and Engineering Research Council of Canada and assistance of S. Dorland are gratefully acknowledged.
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Fermentation inhiblition by acetylene to ammonium in soils. Soil Science Society of America Journal 43, 93 l-936.
Caskey W. H. and Tiedje J. M. (1980) The reduction of nitrate to ammonium by a Closrtidium sp. isolated from soil. Journal of General Microbiology 199, 217-223. de Catanzaro J. B., Beauchamp E. G. and Drury C. F. (1987) Denitrification versus dissimilatory nitrate reduction in soil with alfalfa, straw, glucose and sulfide treatments. Soil Biology & Biochemistry 19, 583-587. Crabbendam P. M., Neijssel 0. M. and Tempest D. W. (1985) Metabolic and energetic aspects of the growth of Closttidium butyticum on glucose in chemostat culture. Archives of Microbiology 142, 375-382.
Daesch G. and Mortenson L. E. (1967) Sucrose catabolism in Closttidium pasreutianum and its relation to N, fixation. Journal of Bacretiology 96, 34&351. Hanawalt R. B. and Steckel J. E. (1967) Ammonium determination by automated distillation from soil suspensions and colored solutions. In Aulomation in Analyrical Chemistry, Technicon Symposium I, pp. 133-I 36. Medied Inc., White Plains, N.Y. Hasan S. M. and Hall J. B. (1975) The physiological function of nitrate reduction in Closttidium petftingens. Journal of General Microbiology 87, 12&128.
Ishimota M., Umeyama M. and Chiba S. (1974) Alteration of fermentation products from butyrate to acetate by nitrate reduction in Closttidiumpetftingens. Zeitschtiftfit Allgemeine Miktobiologie 14, 115-12 1. Jungermann K., Kirchniawy H., Katz N. and Thauer R. K. (1974) NADH, a physiological electron’ donor in clostridial nitrogen fixation. FEBS L&lets 43, 203-206. Jungermann K., -Thauer R. K., Leimonstoil G. and Decker K. (1973) Function of reduced uvridine nucleotide-feriedoxin oxidoreductases in sac&rolytic clostridia. Biochimica and Biophysics Acra 305, 268-280. Kaspar H. F. and Tiedji J. M. (1981) Dissimilatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and the effect of acetylene. Applied and Environmental Microbiology 41, 705-709.
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King D. and Nedwell D. B. (1985) The influence of nitrate concentration upon the end-products of nitrate dissimilation by bacteria in anaerobic salt marsh sediment. FEMS Microbiological Ecology 31, 23-28.
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Knowles R. and Denike D. (1974) Effects of ammonium-, nitrite-, and nitrate-nitrogen on anaerobic nitrogenase activity in soil. Soil Biology & Biochemistry 6, 353-358. Lynch J. M. and Gunn K. B. (1978) Use of a chemostat to study decomposition of wheat straw in soil slurries. Journal of Soil Science 29, 551-556.
Morris J. G. and O’Brien R. W. (1971) Oxygen and clostridia: a review. In Spore Research 1971 (A. N. Barker, G. W. Gould and J. Wolf, Eds). Academic Press, London. Oremland R. S. and Capone D. G. (1988) Use of “specific” inhibitors in biogeochemistry and microbial ecology. In Advances in Microbiological Ecology IO (M. Alexander, Ed.), p. 285. Plenum Press, New York. Oremland R. S., Umberger C., Cubbertson C. W. and Smith R. L. (1984) Denitrification in San Francisco Bay intertidal sediments. Applied and Envitonmenral Microbiology 47, 1106-1112.
Paul J. W. and Beauchamp E. G. (1989a) Rapid extraction and analysis of volatile fatty acids in soil. Communicarions in Soil Science and Planr Analysis 20, 85-94. Paul J. W. and Beauchamp E. G. (1989b) Denitrification and fermentation in plait residueiamended soil. Biology and Fertility of Soils 7, 303-309.
Payne W. J. (1984) Influence of acetylene on microbial and enzymatic assays. Journal of Microbiological Methods 2, 117-133.
Rice W. A. and Paul E. A. (1971) The organisms and biological processes involved in asymbiotic nitrogen fixation in waterlogged soil amended with straw. Canadian Journal of Microbiology 18, 715-723. Smith L. A., Hill S. and Yates M. G. (1976) Inhibition by acetylene of conventional hydrogenase in nitrogen-fixing bacteria. Nature 262, 209-210. Sorensen J. (1978) Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Applied and Environmental Microbiology 35, 301-305.
Thauer R. K., Jungermann K. and Decker K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriological Reviews 41, 100-180.
Watanabe I. and de Guzman M. R. (1980) Effect of nitrate on acetylene disappearance from anaerobic soil. Soil Biology & Biochemisrty 12, 193-194.
Yoshinari T., Hynes R. and Knowles R. (1977) Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biology & Biochemistry 9, 177-183.