Estuarine and Coastal Marine Sdence (x98o) xo, 699--7oz

Notes and Discussions

H e t e r o t r o p h i c N i t r o g e n F i x a t i o n in an I n t e r t i d a l S a l t m a r s h S e d i m e n t

D. B. Nedwell and S. Azni bin Abdul Aziz Department of Biology, University of Essex, Colchester, England

Received x4 ffune I979 and in revised form 2z October z979

Keywords: nitrogen fixation; saltmarsh; sediment; anaerobes; hydrogen; sulphate; sulphur bacteria; England coast

Acetylene reduction was shown to occur under a nitrogen atmosphere in slurries of anaerobic saltmarsh sediment. This acetylene reduction was entirely inhibited by 2o n~f molybdate, but was stimulated by the presence of a hydrogen atmosphere. Anaerobic heterotrophie nitrogen fixation was, therefore, attributed to the activity of sulphate reducing bacteria which were the predominant nitrogen fixers in this environment.

Introduction

The presence of heterotrophie nitrogen fixation in the absence of light has been demon- strated in a variety of situations including marine sediments (Patriquin & Knowles, x97z; Herbert, x975). A number of bacteria with the capability of fixing nitrogen have been isolated in laboratory culture from a marine sediment. Herbert (I975) suggested that in an anaerobic marine sediment of the Tay estuary Desulfovibrio spp. were probably the most important heterotrophic nitrogen fixing bacteria, as indicated by the salt requirement of their nitrogenase enzymes and their presence in high numbers. Most other Nz fixing heterotrophs appeared to be intrusive freshwater forms, or present ortly in low numbers.

In the Colne Point saltmarsh on the Essex coast high rates of nitrogen fixation have been demonstrated, the greatest rate being zx. 4 lag atoms N~ fixed per square centlmetre of sediment per year in a saltmarsh pan (Abdul Aziz & Nedwell, x979); of this annual total, hetcrotrophie dark fixation represented xx. 7 lag atoms N2 cm -2 year -x (Abdul Azlz, i979). The present work was undertaken to determine whether the sulphate reducing bacteria were the predominent heterotrophie nitrogen fixers in this environment. Sediment was obtained from a saltmarsh pan described by Nedwell & Abram (x978), with a benthic mat of Oscillatoria limosa. The sediment was highly reduced and sulphate reduction was active.

Methods

Sediment was removed from the surface 5 cm of the saltmarsh pan, the zone previously demonstrated (Nedwell & Abram, x978 ) to have the greatest microbial activity. A 50% (v/v) slurry of sediment was made by addition of seawater which had previously been deoxygenated by bubbling through oxygen-free nitrogen for 3 ° mln. Aliquots of sediment

699 o3oz-3524/8o[o6o699+o4 $o2.oo/o © x98o Academle Press Inc. (London) Ltd.

700 D. B. Nedwell 6Y S. A . bin Abdul A z i z

(5 ° ml) were dispensed into I5O ml conical flasks sealed with Subaseals, and the flasks were immediately gassed out with either Nz/CO 2 (8o% : 2o%, v/v) or HJCO2 (8o% : 2o%, v/v1. A pressure of o.2 atm of acetylene gas was then added to each flask (Hardy et al., I97I ).

Sodium molybdate solution was added to duplicate flasks under each gas mixture to give a final concentration of 2o m~t molybdate. An equivalent volume of seawater was added to a second duplicate set of flasks. The flasks were then shaken on a rotary shaker (I6o cycles rain-l) at a temperature of 2o °C in the dark. Samples of the gas atmosphere in each flask were periodically removed and assayed for their ethylene content in a Perkin-Elmer F33 gas, chromatograph (Poropak N column, 8o °C, nitrogen carrier gas)

Results

The results of the experiment are shown in Figure z.

I i l i I 1

I 00

_..-. I

5o

£ ,Y

0 I 2 3 4 5 6 7

Time {doys)

Figure I. The reduction of acetTlene by slurries of anaerobic saltmarsh sediment. O, N,/CO= atmosphere; O, N,/COzWmolybdate (2o re.x0; 1-3, H,/CO, atmosphere =, H,/CO,+molybdate (20m.~0. Bars indicate spread,of analyses in duplicate flasks

Discussion

The results showed that dark heterotrophic nitrogen fixation occurred in the sediment sample under N2/CO ~ and that fixation was entirely inhibited by the presence of 2o m~t molybdate. Oremland & Taylor (I978) have shown molybdate to be a selective inhibitor of

Nitrogen fixation in a saltmarsh sediment 7Ol

sulphate reducing bacteria, an observation supported by data in our own laboratory (D. B. Nedwell, unpublished data). Its mode of action is presumablydue to the chemical similarity of sulphur and other elements of Group VI in the periodic table such as molybdenum, selenium, tellurium or chromium. Postgate (x952) and Saleh et al. (x964) have shown selenate, tellurate and chromate to be inhibitory to sulphate reducing bacteria, and Taylor & Oremland (I977) reported that molybdate depleted ATP levels in whole cells of Desulfoz;ibrio. This is con- sistent with molybdate acting as art inhibitory analogue of sulphate in the electron transport mechanism of the sulphate reducers. Our results indicate, therefore, that heterotrophie nitrogen fixation in this anaerobic intertidal sediment was virtually entirely due to the activity of the sulphate reducing bacteria, supporting the conclusion of Herbert (x975).

Moreover, Patriquin & Knowles (x972) and Herbert (x975) have demonstrated that heterotrophic nitrogen fixation was limited by the supply of electron donor available to the nitrogen fixing heterotrophs in marine sediment. The stimulation of acetylene reduction induced by the presence of hydrogen again supports this conclusion as Abram & Nedwell (x978) have demonstrated that hydrogen is an important electron donor for sulphate reducing bacteria in saltmarsh sediment. Patriquin & Knowles (x975) also reported the stimulation of acetylene reduction by hydrogen in a carbonate sand and attributed this to the activity of sulphate reducing bacteria. Hydrogen is a competitive inhibitor of nitrogen fixation but not of acetylene reduction (Hwang et al., x973). However, in the natural sediment the concentra- tion of hydrogen will be maintained at a low concentration by the hydrogen scavenging activity of sulphate reducing bacteria and methanogenic bacteria and will not, therefore, be expected to inhibit the hz situ activity of nitrogen fixing bacteria.

The enhancement of the rate of acetylene reduction under hydrogen in the presence of molybdate suggests that in the presence of abundant electron donor the activity of other groups of hydrogen utilizing, nitrogen fixing anaerobes becomes more significant Certain Clostridlum spp. are known to be capable of fixing nitrogen and some are also capable of utilizing hydrogen (e.g. Ohwaki & Hungate, x977). However, in the unamended natural sediment the use of molybdate as a selective inhibitor of sulphate reducing bacteria showed them to be the predominant nitrogen fixing bacteria.

References

Abdul Aziz, S. A. bin x979 A study of nitrogen cycling in a coastal saltmarsh ecosystem. Ph.D. thesis, University of Essex.

Abdul Aziz, S. A. bin & Nedwell, D. B. x979 Microbial nitrogen transformations in the salt marsh environment. In Ecological Processes in Coastal Environments (Jefferies, R. L. & Davy, A. J., eds). Blackwell Scientific Publications, Oxford, pp. 385-398.

Abram, J. ~V. & Nedwell, D. B. I978 Hydrogen as a substrate for methanogenesis an d sulphate reduc- tion in anaerobic saltmarsh sediment. Archives of Microbiology II7~ 89-92.

Hardy, R. XV. F., Burns, R. C., Herbert, R. R., Holsten, R. D. & Jackson, E. K. x97x Biological nitrogen fixation: a key to world protein. Plant Science Special Volume, 56x-59o.

Herbert, R. A. I975 Heterotrqphie nitrogen fixation in shallow estuarine sediments.ffournal of Experi- mental J~farine Biology and Ecology ISj 2t5-225.

Hwang, J. C., Chen, C. H. & Burriss, R. H. t973 Inhibition of nitrogenase-catalyzed reductions. Biochhnlca et Biophysica Acta z92, z56-27o.

Nedwell, D. B. & Abram, J. W. I978 Bacterial sulphate reduction in relation to sulphur geochemistry in two contrasting areas of saltmarsh sediment. Estuarine and Coastal Marine Science 6~ 34t-35x.

Ohwaki, K. & Hungate, R. E. x977 Hydrogen utilisation by clostridia in sewage sludge. Applied and Environmental Microbiology 33, xz7o-t274.

Oremland, R. S. & Taylor, B. F. x978 Sulfate reduction and methanogenesis in marine sediments. Geochimica et Cosmochimica Acta 42, 2o9-2z4.

Postgate, J. R. x952 Competitive and non-competitlve inhibitors of bacterial sulphate reduction. Journal of General 2~ficrobiology 6, x28-x42

7oz D. B. Nedzcell & S. A. bin Abdul Azlz

Patriquin, D. & Knowles, R. x97z Nitrogen fixation in the rhizosphere of marine angiosperms. Marine Biology x6j 49-$8.

Patriquin, D. & Knowles, R. x975 Effects of oxygen, mannitol and ammonium concentrations on nitrogenase (C2H2) activity in a marine skeletal carbonate sand. Marine Biology 32, 49-62.

Saleh, A. M., MacPherson, R. M. & Miller, J. D. A. x964 The effect ofinhibitors on sulphate-reducing bacteria: a compilation. ~ournal of Applied Bacteriology 27, 28X--293

Taylor, B. F. & Oremland, R. S. x 977 Specific depletion of cellulose ATP in Desulfovibrlo by molybdate and other Group VI anions. Annual Meeting of the American Society for/~Iicrobiology, Abstract Q4x, pp. 068.