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Anomalous results encountered when measuring acetylene reduction by soil bacteria
Soit &of. Bro&m
Vol 7. pp. 403-404
Pergamon
Press 1975. Printed :n Great Britam.
SHORT COMMUNISATION Anomalous results encountered when measuring acetylene reduction by soil bacteria P. M. MURPHY Department of Soil Biology, Agricultural Institute, Johnstown Castle, Wexford, Ireland
This report describes the reduction of C,H, to C,H, and C,H, on an inorganic catalyst by HZ evolved from anaerobic decomposition of plant root material. The presence of transition metals and H, gave highly erroneous results when estimating activities of C,H2 reducing bacteria in soils, and the main interest of this report is that it further demonstrates the need for caution when using the CZH, reduction method as a measure of N? fixation. When assessing the activity of N, fixing anaerobes in soil, using C,H, reduction to measure nitrogenase activity, (Hardy et al., 1968) modified McIntosh and Fildes pattern anaerobic cuiture jars (~allenkamp, London) were tested as suitable incubation chambers. Certain results obtained in these assays suggested that a system, other than the biological one, was also active in reducing &Hz to C,H,. As these jars are fitted with a catalyst. which aids in producing anaerobic conditions, it was considered likely that this was also catalyzing the non-enzymic reduction of CIH2. Control assays run in air or anaerobi~lly in N, in which C,H2 (6.65 mmoles) only was contained in the incubation chamber, showed no C,H, formation. However, inclusion of soil cores obtained from barley field plots resulted, after an incubation period of 2448 h, in considerable C,H4 formation under anaerobic conditions (Fig. 1). There was no CzHn formation under aerobic conditions. Disruption of the soil core and removal of the root material also resulted in insi~ifi~nt C,H, formation, and finally, on removal of the catalyst from the jar, there was no C,H, reduction by the intact soil core under anaerobic conditions. The effect of various H, concentrations, on the formation of C,H, from C,H, by the catalyst, show clearly (Table 1) that this catalyst material is very effective in promoting the reduction of C,H, by Hz to form C,H,. As aerobic assays showed a 50% reduction in activity, assays
Fig. I. Time curve for C,H, reduction on the inorganic catalyst in the presence of endogenous H,. Assayed in McIntosh and Fildes jar (3 1.) containing soil cores (two 150 x 75 mm, 9OOgwet wt.) in 1 atm Nz and 6.65 mmol C,H,, incubated at 20°C. Results are the mean of triplicate estimations.
Table 1. Formation of C,H, from C&H,, on the inorganic catalyst, at various H, concentrations pmoles Hz added
/imoles C,H, produced
8,x* 17.6 264 35.2
44 9.0 142 22-o
“i H, recovery as C,H4 SO.0 51.0 53.7 62.5
* Results are the mean of triplicate estimations. were carried out anaerobically in 30 ml McCartney bottles containing the catalyst (20 beads/bottle) in I.0 atm N, and C2H, (133 jlmoles) at 20°C and HZ at various concentrations. After 1 h incubation 0.2 ml of the gas phase was removed and the CZHZ/C,H, ratio measured by a HZ-flame ionisation detector on a Perkin-Elmer model F I1 gas chromatograph. The gases were separated on a 1.2 m x 2 mm column of Porapak N at a temperature of 50°C using N2 as carrier gas (Roughley and Dart, 1969). Under these conditions the C,H, concentration was saturating and the H, concentrations were in all cases limiting. As may be seen from Table 1, C,H, recovery values, based on Hz concentrations used varied from 50 to 62%. An upward trend in CZHe recovery with increasing Hz concentration is evident. Results below suggest that the reason why recoveries of only approximately 50% are obtained is due to the binding of some of the C2H4 formed on the catalyst, where it is further reduced to C2H,. At equimolar concentrations of CZH2/H, (44 pmoles in 1.0 atm N,) a large CzHs peak (78 s) emerges after the C,H, peak (68 s) and before the CzHz peak (124 s). Under these experimental conditions the gases are defected in a final ratio of C,H,:C,H,:C,H, of 2,5:3:1. At a C,H,/H, concentration ratio of l-3 there is a complete co&e&on of C,H2 to C2H, and C,H, which are formed in a ratio of 1: 3 respectively. This further reduction step leading to the formation of&H, is in sharp contrast with the highly specific biological system where C,H, is the final end product formed, there being no instance recorded of any further reduction to C2H6. As in the enzyme system, CO inhibits the reduction of C,H2 on the inorganic catalyst. Figure 2 shows the time curve for the reduction of C,H, on the catalyst, at two H, concentrations when the CzHz was present in excess. The initial rate of C,H4 formation is greater at the higher H, concentration, the reaction being 50% complete in just over 90 s [Fig. 2(b)]; the corresponding time at the lower H, concentration is almost 150 s [Fig. 2(a)]. The amount of C,H, recorded is again proportionately greater at the higher H, concentration in agreement with the results shown in Table 1. It appears therefore that under the experimental conditions in Fig. I, anaerobic biological activity in the soil
403
Short communications
404
mediating the reduction of these gases and palladium. platinum and iron are the primary metals contained in the inorganic catalyst investigated here. It is interesting to note the production of considcrahle quantities of HL bq the soil cores (~Oilrnol H?g-’ soil 4X hh-- Fig. I), in view of the ease with which this gas reduces CLHz in the presence of transition metals. Also, although this evolution of H, in closed assay systems will not inhibit biological C,H, reduction, accumulation of Hz would be expected to inhibit N, fixation I)L’~ se (Hwang and Burris. IYhX).
Seconds
Seconds
Fig. 2. Time curve for CZH, reduction on inorganic catalyst in presence of exogenous H,. Assayed in McCartney hottlc (30 ml) containing 1 atm N:. 44.0 Ltmoles CLH2 and (a) 088 pmoles H, (b) 8.X pmoles Hz at 20 C. Results are the mean of triplicate estimations.
cores produced H, which, in the presence of C2HZ and the catalyst, resulted in the formation of the CZH, concentrations measured. The abiological reduction of Nz and C,H, under mild conditions is well documented and was recently reviewed (Hardy et trl.. I Y73). The transition metals have been shown to bc parttcularly ctfectivc in
REFERENCES HARDY R. W. F.. H~LSTEN R. D., JACKSON E. K. and BLKUS R. C. (1968) The acetylene-ethylene assay for nitrogen fixation. PI. Physiol. 43, I 1x5 1207. HARDY R. W. F., BURNS R. C. and PARSHALL G. W. (1973) Bioinorganic chemistry of dinitrogen fixation. In Itrorgatric ~jo~~7~,~~is~r~pp. 745.-793 (G. Eicllhorn, Ed.) Elsevicr. Amsterdam. HWANG J., and RURRIS R. H. (1968)Binding sites of nitrogenase. Ftdrratior~ Proc. 21, 639. ROLGHLEY R. J.. and DART P. J. (1969) Reduction of acctylcne by nodules of Trifi~litnrt .suhrer,nrrrurn as affected by root temperature. Rhixhiunl strain. and host culttvar. A&. .&fikrohid. 69, 17 I- 179.
Vol 7. pp. 403-404
Pergamon
Press 1975. Printed :n Great Britam.
SHORT COMMUNISATION Anomalous results encountered when measuring acetylene reduction by soil bacteria P. M. MURPHY Department of Soil Biology, Agricultural Institute, Johnstown Castle, Wexford, Ireland
This report describes the reduction of C,H, to C,H, and C,H, on an inorganic catalyst by HZ evolved from anaerobic decomposition of plant root material. The presence of transition metals and H, gave highly erroneous results when estimating activities of C,H2 reducing bacteria in soils, and the main interest of this report is that it further demonstrates the need for caution when using the CZH, reduction method as a measure of N? fixation. When assessing the activity of N, fixing anaerobes in soil, using C,H, reduction to measure nitrogenase activity, (Hardy et al., 1968) modified McIntosh and Fildes pattern anaerobic cuiture jars (~allenkamp, London) were tested as suitable incubation chambers. Certain results obtained in these assays suggested that a system, other than the biological one, was also active in reducing &Hz to C,H,. As these jars are fitted with a catalyst. which aids in producing anaerobic conditions, it was considered likely that this was also catalyzing the non-enzymic reduction of CIH2. Control assays run in air or anaerobi~lly in N, in which C,H2 (6.65 mmoles) only was contained in the incubation chamber, showed no C,H, formation. However, inclusion of soil cores obtained from barley field plots resulted, after an incubation period of 2448 h, in considerable C,H4 formation under anaerobic conditions (Fig. 1). There was no CzHn formation under aerobic conditions. Disruption of the soil core and removal of the root material also resulted in insi~ifi~nt C,H, formation, and finally, on removal of the catalyst from the jar, there was no C,H, reduction by the intact soil core under anaerobic conditions. The effect of various H, concentrations, on the formation of C,H, from C,H, by the catalyst, show clearly (Table 1) that this catalyst material is very effective in promoting the reduction of C,H, by Hz to form C,H,. As aerobic assays showed a 50% reduction in activity, assays
Fig. I. Time curve for C,H, reduction on the inorganic catalyst in the presence of endogenous H,. Assayed in McIntosh and Fildes jar (3 1.) containing soil cores (two 150 x 75 mm, 9OOgwet wt.) in 1 atm Nz and 6.65 mmol C,H,, incubated at 20°C. Results are the mean of triplicate estimations.
Table 1. Formation of C,H, from C&H,, on the inorganic catalyst, at various H, concentrations pmoles Hz added
/imoles C,H, produced
8,x* 17.6 264 35.2
44 9.0 142 22-o
“i H, recovery as C,H4 SO.0 51.0 53.7 62.5
* Results are the mean of triplicate estimations. were carried out anaerobically in 30 ml McCartney bottles containing the catalyst (20 beads/bottle) in I.0 atm N, and C2H, (133 jlmoles) at 20°C and HZ at various concentrations. After 1 h incubation 0.2 ml of the gas phase was removed and the CZHZ/C,H, ratio measured by a HZ-flame ionisation detector on a Perkin-Elmer model F I1 gas chromatograph. The gases were separated on a 1.2 m x 2 mm column of Porapak N at a temperature of 50°C using N2 as carrier gas (Roughley and Dart, 1969). Under these conditions the C,H, concentration was saturating and the H, concentrations were in all cases limiting. As may be seen from Table 1, C,H, recovery values, based on Hz concentrations used varied from 50 to 62%. An upward trend in CZHe recovery with increasing Hz concentration is evident. Results below suggest that the reason why recoveries of only approximately 50% are obtained is due to the binding of some of the C2H4 formed on the catalyst, where it is further reduced to C2H,. At equimolar concentrations of CZH2/H, (44 pmoles in 1.0 atm N,) a large CzHs peak (78 s) emerges after the C,H, peak (68 s) and before the CzHz peak (124 s). Under these experimental conditions the gases are defected in a final ratio of C,H,:C,H,:C,H, of 2,5:3:1. At a C,H,/H, concentration ratio of l-3 there is a complete co&e&on of C,H2 to C2H, and C,H, which are formed in a ratio of 1: 3 respectively. This further reduction step leading to the formation of&H, is in sharp contrast with the highly specific biological system where C,H, is the final end product formed, there being no instance recorded of any further reduction to C2H6. As in the enzyme system, CO inhibits the reduction of C,H2 on the inorganic catalyst. Figure 2 shows the time curve for the reduction of C,H, on the catalyst, at two H, concentrations when the CzHz was present in excess. The initial rate of C,H4 formation is greater at the higher H, concentration, the reaction being 50% complete in just over 90 s [Fig. 2(b)]; the corresponding time at the lower H, concentration is almost 150 s [Fig. 2(a)]. The amount of C,H, recorded is again proportionately greater at the higher H, concentration in agreement with the results shown in Table 1. It appears therefore that under the experimental conditions in Fig. I, anaerobic biological activity in the soil
403
Short communications
404
mediating the reduction of these gases and palladium. platinum and iron are the primary metals contained in the inorganic catalyst investigated here. It is interesting to note the production of considcrahle quantities of HL bq the soil cores (~Oilrnol H?g-’ soil 4X hh-- Fig. I), in view of the ease with which this gas reduces CLHz in the presence of transition metals. Also, although this evolution of H, in closed assay systems will not inhibit biological C,H, reduction, accumulation of Hz would be expected to inhibit N, fixation I)L’~ se (Hwang and Burris. IYhX).
Seconds
Seconds
Fig. 2. Time curve for CZH, reduction on inorganic catalyst in presence of exogenous H,. Assayed in McCartney hottlc (30 ml) containing 1 atm N:. 44.0 Ltmoles CLH2 and (a) 088 pmoles H, (b) 8.X pmoles Hz at 20 C. Results are the mean of triplicate estimations.
cores produced H, which, in the presence of C2HZ and the catalyst, resulted in the formation of the CZH, concentrations measured. The abiological reduction of Nz and C,H, under mild conditions is well documented and was recently reviewed (Hardy et trl.. I Y73). The transition metals have been shown to bc parttcularly ctfectivc in
REFERENCES HARDY R. W. F.. H~LSTEN R. D., JACKSON E. K. and BLKUS R. C. (1968) The acetylene-ethylene assay for nitrogen fixation. PI. Physiol. 43, I 1x5 1207. HARDY R. W. F., BURNS R. C. and PARSHALL G. W. (1973) Bioinorganic chemistry of dinitrogen fixation. In Itrorgatric ~jo~~7~,~~is~r~pp. 745.-793 (G. Eicllhorn, Ed.) Elsevicr. Amsterdam. HWANG J., and RURRIS R. H. (1968)Binding sites of nitrogenase. Ftdrratior~ Proc. 21, 639. ROLGHLEY R. J.. and DART P. J. (1969) Reduction of acctylcne by nodules of Trifi~litnrt .suhrer,nrrrurn as affected by root temperature. Rhixhiunl strain. and host culttvar. A&. .&fikrohid. 69, 17 I- 179.