- Email: [email protected]
Nitrogen fixation by Azotobacter chroococcum in the presence of Colpoda steini—II. The influence of agitation
Soil Biol. Biochem. Vol. 4, pp. 371-376. Pergamon Press 1972. Printed in Great Britain
NITROGEN FIXATION BY AZOTOBACTER CHROOCOCCUM IN THE PRESENCE OF COLPODA STErNI-II. THE INFLUENCE OF AGITATION J. F.
DARBYSHIRE
The Macaulay Institute for Soil Research, Craigiebuckler, (Accepted
Aberdeen
3 May 1972)
Summary-The
effects of agitation on the growth and N, fixation of pure cultures of Azotobacter chroococcum Beijerinck strain 7115 and mixed cultures of A. chroococcum strain 7115 with Colpodu steini Maupas are reported. The liquid cultures were incubated at 28°C and an
orbital shaking machine was used at 60 rev/min. In the first 14 days of incubation all the shaken cultures fixed significantly more N, than the static cultures (PC 0.01). Nz fixation in the static cultures in the third and fourth weeks of incubation surpassed the total quantities of N, fixed in the shaken cultures. The quantities of Nz fixed by mixed and pure cultures of Azotobucter sampled on the same day were not significantly different (PC 0.05) in either the static or shaken cultures. There were fewer Azotobacter cells present in mixed cultures compared with pure Azotobucter cultures sampled at the same time, when trophic ciliates were present. The differences in the size of Azotobacter populations between the mixed and pure cultures were less marked amongst the shaken than in the static cultures. The normal morphological changes of Azotobocter cells associated with increasing population age did not appear to be delayed by ciliate predation. Also, there was no evidence to show that ciliate predation influenced the proportion of viable cells in the total Azofobucter populations. INTRODUCTION THE EXPERIMENTAL results reported in the first paper (Darbyshire, 1972) in this series confirmed earlier reports of increased nitrogen fixation by Azotobacter chroococcum Beijerinck in the presence of a soil ciliate, Colpoda steini Maupas, when these microbes were incubated at temperatures between 15 and 25°C. In similar experiments at 28”C, the mixed cultures of Azotobacter and Colpoda frequently fixed less nitrogen than the respective pure cultures of Azotobacter. In view of the known oxygen sensitivity of Azotobacter during nitrogen fixation (Parker, 1954, Posgtate, 1971), it was suggested that the ciliates may help to reduce the dissolved oxygen concentrations in the mixed cultures of Azotobacter and Colpoda to more suitable levels for nitrogen fixation between 15 and 25°C. At 28°C the respriatory activity of Azotobacter may be sufficiently large to reduce the dissolved oxygen content to suitable levels in the absence of ciliates. Also, the solubility of oxygen in aqueous media is less at 28°C than at these lower temperatures. This paper reports the results of experiments with shaken and static cultures of Azotobacter chroococcum strain 7115 incubated at 28°C in the presence and absence of Colpoda steini. These experiments were designed as a partial test of the above hypothesis. MATERIALS
AND METHODS
The origin of the microbes and methods of counting the total number of cells were the same as described by Darbyshire (1972). The trophic Colpoda were only counted in cultures that had been incubated for 17, 24 or 31 days, although trophic Colpoda were observed in 371
372
J. F. DARBYSHIRE
the culture flasks on days 3, 10 and 14. The viable numbers of Azotobacter were counted by the drop count method as described by Collins (1964), except that ten drops from each dilution were used. The Azotobacter colonies were counted with an inverted microscope (Zeiss, Oberkiichen) after they had been incubated at 25°C for 2 days. The nutrient agar used (BBJ medium) was the medium described by Brown, Burlingham and Jackson (1962), except that K,HP04 was not sterilized separately. The diluent in the drop count method was BBJ medium without agar or glucose. The liquid medium used in the experiments was the BBJ medium without agar. The cultures were incubated at 28°C either unshaken in an incubator or shaken on an orbital shaker (New Brunswick Scientific Co. Model G.25) at 60 rev/min. The other experimental details were the same as described by Darbyshire (1972). RESULTS
The total quantities of nitrogen and carbon detected in mixed or pure cultures of A chroococcum 7115, when the flasks were incubated unshaken at 28”C, are shown in Table 1. TABLE 1. TOTAL NITROGEN &g/ml) AND TOTAL CARBON (mg/ml) IN PURE A. chroococcum STRAIN 7115 AND MIXED A. chroOCOCCU~STRAIN 7115/C. steini CULTURES INCUBATED AT 28°C and UNSHAKEN
Days after inoculation
A. chroococcum
N
C A. chroococcum and C. steini
N C
14
21
25
28
16 1.5*
37 1.4
43 0.8
51 0.6
11
34 1.3
38 0.9
53 0.5
1.3
* Significantly different (PC 0.05) from the carbon the mixed culture and sampled on the same day.
value in
The static mixed cultures of Azotobacter and Colpoda fixed less nitrogen than the static pure Azotobacter cultures on days 14,21, 25 and 28 (not significant, P
NITROGEN
FIXATION
BY AZOTOBACTER
’ 10 Days
after
1-41:
CHROOCOCCUM
24
373
31
inoculation
FIG. 1. Microbial populations in pure A. chroococcum strain 7115 and mixed A. chroococcunz strain 7115/C. steini cultures incubated unshaken at 28°C. White circles (0) and squares (0) refer to the total and viable Azotobacter populations in pure cultures. Black circles (a), squares (H), diamonds (+) and triangles (N refer to the total and viable Azotobacter populations in mixed CoZpodu/Azorobacter cultures, to the total ciliate populations and to the trophic ciliate populations respectively.
cocci with refractile granules were observed on day 24 in both pure and mixed cultures. At the end of the experiment on day 31, some of the Azotobacter cells in the pure cultures and most Azotobacter cells in the mixed cultures were cysts. A dark pigment was first observed in the majority of the pure cultures on day 21 and in most of the mixed cultures the same pigment appeared on day 22. Subsequently, the pure and mixed cultures were indistinguishable in terms of colour. The chemical analyses (Table 2) from a similar experiment with shaken Azotobacter cultures showed that the shaken mixed cultures fixed an extra 5 pg N/ml more than the shaken pure cultures in the first 2 weeks (not significant, P-C 0 -05). After longer periods of incubation the quantities of nitrogen were almost identical in both pure and mixed shaken cultures. A comparison between all the static and shaken cultures showed that all the static cultures fixed significantly less nitrogen than the shaken cultures in the first 2 weeks (PC O-01) but when the incubation period was for 21 days or longer the nitrogen fixation in the static cultures surpassed significantly the shaken cultures (P < 0.01). The microbial populations of the shaken cultures (Fig. 2) exhibited several differences from that described for static cultures (Fig. 1). The shaken Azotobacter populations in both pure and mixed cultures multiplied more rapidly than the equivalent static cultures in the first 10 days of incubation (significant at PcO.01). The total and viable Azotobacter populations in the shaken pure cultures were significantly larger than the equivalent Azotobacter S.B.B. 413-H
J. F. DARBYSHIRE
374
TABLE 2. TOTAL NITROOEN&g/ml) AND TOTALCARBON(mg/ml) IN PURE A. chroococcum STRAIN 7115 AND MIXED A. chroococcum STRAIN 7115/C. steini CULTURESINCUBATEDAT 28°C AND SHAKENAT 60 rev/min
Days after inoculation
A. chroococarm
A. chroococcum
and C. steini
14
21
25
28
C
18 0.9
25 l.O*
27 0.9
26 0.8
N C
23 0.9
26 0.8
26 0.8
27 0.7
N
* Significantly different (PcO.05) from the carbon value in the mixed culture and sampled on the same day.
FIG. 2. Microbial populations in pure A. chroococcum strain 7115 andmixed A. chroococcum strain 7115/C. steini cultures incubated at 28°C and shaken at 60 rev/min. White circles (0) and squares (0) refer to the total and viable Azotobucter populations in pure cultures. Black circles (O), squares (m), diamonds (+) and triangles (m refer to the total and viable Azotobacter populations in mixed Cofpodu/Azotobucter cultures, to the total ciliate populations and to the trophic ciliate populations respectively.
NITROGEN FIXATION BY AZOTOBACTER
CHROOCOCCUM
375
populations found in shaken mixed cultures sampled on the same day (P < 0 -01) except for total populations on day 4, 17 and 24. These differences in Azatobacter populations between mixed and pure shaken cultures were, however, generally smaller than equivalent differences in Azutobaeter populations described earlier for pure and mixed static cultures. Total ciliate populations in shaken cultures were also much smaller than the total ciliate populations found in static cultures from day 3 onwards (significant at P < O-001). Trophic Colpou’u were observed in the shaken flasks from day 3 onwards and fewer individuals were observed after day 10. The morphological changes in the shaken Azoto~~~te~ populations occurred more rapidly than in the static cultures. Many coccoid rods and some cysts were present in shaken pure and mixed cultures on day 10. The majority of Azotobacter cells in both shaken mixed and pure cultures on day 18 were cocci with refractile granules frequently surrounded by mucilage. Both shaken pure and mixed cultures by day 24 contained large numbers of precystic cocci with refractile granules and cysts. A few of the shaken mixed or pure Azotob~cte$ cultures began to develop a dark pigment on day 4 and most of the shaken flasks had a dark colour by day 7. The shaken pure Azotobacter cultures were slightly darker than shaken mixed cultures of the same age until after day 22 when they were indistinguishable in colour. DI!S&USSiON
A comparison between the chemical analyses for shaken and static cultures of A. chro~COCCU~(Tables 1 and 2) suggest that nitrogen fixation and carbon loss were more rapid in the shaken cultures in the first 2 weeks of incubation. In the third and fourth weeks of incubation there was much less nitrogen fixation in the shaken flasks, especially in the mixed cultures. The microscopical observations of the shaken cultures showed that during the latter 2 weeks there was an increasing proportion of cysts and precystic cocci in the Azotobacter populations and a relative scarcity of actively dividing rods. Jensen (1954) concluded that nitrogen fixation is usually associated with cell proliferation and it would appear that the results from the present shaken experiment support this view. Similarly, the more gradual rate of nitrogen fixation and carbon loss in the static cultures was associated with a slower rate of increase in size of the Azotobacter populations and with the presence of some Azotobacter rods throughout the experiment. If the suggestion that the ciliates are able to maintain Azotobacter cells in a ‘ . . . younger and more active condition. . . ’ is correct (Cutler and Bal 1926), then it might be expected that Azotobacter cysts would be less frequent and rods more frequenr in mixed shaken cultures in the first 2 weeks than in shaken pure Azotobacter cultures of the same age. Although the numbers of Azotobacter cysts were not counted, the shaken mixed cultures did not appear to have a smaller proportion of Azotobacter cysts or a larger proportion of rods in the total bacterial population during the first 2 weeks of incubation compared with shaken pure cultures. The relationship between the presence of trophic Colpoda and smaller populations of Azotobacter in mixed compared with pure cultures previously reported (Darbyshire, 1972), is not so well defined in the present shaken experiment. The small differences between Azotobffcter populations in shaken mixed and pure cultures may be due to the small numbers of trophic Colpodu present in many of the shaken cultures as well as to the more rapid multiplication of Azotobacter in the shaken compared with the static cultures. There was no indication in the shaken or static experiment that ciliate predation affects the viability of the Azotobacter populations. It is still possible, however, that ciliate predation may influence
376
3. F. DARBYSHIRE
the rate of decline of the viable count of Azotobacter in older cultures, which had reachec the so-called growth phase of decline (Cooper, 1971). The much lower total Colpoda populations from day 3 onwards in the shaken compared with the static cultures were associated with the accelerated onset of Azotobacter cyst formation and dark pigmentation in the shaken cultures. Further experiments are in progress to determine whether Azotobacter cysts are intrinsically inedible for Colpoda or whether the dark pigment renders them unsuitable for food. As some of this pigment appears to be released into the mixed culture media, it is possible that such pigments might be released from Azotobacter cells in the soil and discourage Colpoda from devouring adjacent edible food. If this phenomenon has a more general appIjcation to soil protozoa, then such microbial pigments may significantly delay the decomposition of microbial cells in the soil. Any conclusions about the feeding habits of soil protozoa based entirely on monoxenic bacterial/protozoan cultures would also have to be modified. The shaking treatment is Iikely to affect microbial growth and metaboi~sm in several ways besides improving the oxygen transfer rate. Future measurements of the dissolved oxygen concentrations in pure and mixed Azofobacter cultures should provide more direct evidence that protozoa can reduce the dissolved oxygen concentration of Azotobacter cultures significantly. Nevertheless, the results of two experiments described previously (Darbyshire, 1972), showed that Cofpodu steini stimulated nitrogen fixation by Azotobacter to a greater extent when mixed cultures of 100 ml aliquots of unshaken culture fluid in 1 I. Rasks were used, rather than 40 ml aliquots of the same unshaken medium in 150 ml flasks. It is assumed that the oxygen transfer rate in the 1 1. flask experiment was superior to the condition that developed in the smaller flasks. Ack~ow~edgeme~f-the carbon and nitrogen.
author wishes to thank Dr G. ANDERSONwho analysed the culture media for total
REFERENCES BROWN M. E., BURUNGHAM S. K. and JACKSONR. M. (1962) Studies on Azofobacter species in soil---I.
Comparison
of media and techniques for counting Azorobacter in soil. PI. Soil 17, 309-319.
COLLINSC. H. (1964) Microbial Methods, Butterworths, London. COOPERK. E. (1971) Growth of micro-organisms in artificial culture. In Micro-organ&z.r
Fcmction, Form and Environment (L. E. Hawker and A. H. Linton, Eds), pp. 169-192, Arnold, London. CUTLERD. W. and BAL D. V. (1926) In~uence of protozoa on the process of nitrogen fixation by Azofobucfer cbroococcum. Ann. cippl. Biol. 13,516-534. DARBYSHIRE J. F. (1972) Nitrogen fixation by Azofobucfer chroococcum in the presence of Colpodu steini-I. The influence of temperature. Soil Biol. Biochem. 4,359-369. JENSENH. t. (1954) The Azotobacteriaceae. Bucf. Rev. 18, 195-214. PARKERC. A. (1954) Effect of oxygen on the fixation of nitrogen by Azotobacter. Nature, Land. 173,780-781. POSTGATEJ. (1971) Relevant aspects of the physiological chemistry of nitrogen fixation, In ~jcrobe~ and 3iolog~col Prod~cfiv~ty (D. E. Hughes and A. H. Rose, Ed& pp. 287-307, Cambridge University Press.
NITROGEN FIXATION BY AZOTOBACTER CHROOCOCCUM IN THE PRESENCE OF COLPODA STErNI-II. THE INFLUENCE OF AGITATION J. F.
DARBYSHIRE
The Macaulay Institute for Soil Research, Craigiebuckler, (Accepted
Aberdeen
3 May 1972)
Summary-The
effects of agitation on the growth and N, fixation of pure cultures of Azotobacter chroococcum Beijerinck strain 7115 and mixed cultures of A. chroococcum strain 7115 with Colpodu steini Maupas are reported. The liquid cultures were incubated at 28°C and an
orbital shaking machine was used at 60 rev/min. In the first 14 days of incubation all the shaken cultures fixed significantly more N, than the static cultures (PC 0.01). Nz fixation in the static cultures in the third and fourth weeks of incubation surpassed the total quantities of N, fixed in the shaken cultures. The quantities of Nz fixed by mixed and pure cultures of Azotobucter sampled on the same day were not significantly different (PC 0.05) in either the static or shaken cultures. There were fewer Azotobacter cells present in mixed cultures compared with pure Azotobucter cultures sampled at the same time, when trophic ciliates were present. The differences in the size of Azotobacter populations between the mixed and pure cultures were less marked amongst the shaken than in the static cultures. The normal morphological changes of Azotobocter cells associated with increasing population age did not appear to be delayed by ciliate predation. Also, there was no evidence to show that ciliate predation influenced the proportion of viable cells in the total Azofobucter populations. INTRODUCTION THE EXPERIMENTAL results reported in the first paper (Darbyshire, 1972) in this series confirmed earlier reports of increased nitrogen fixation by Azotobacter chroococcum Beijerinck in the presence of a soil ciliate, Colpoda steini Maupas, when these microbes were incubated at temperatures between 15 and 25°C. In similar experiments at 28”C, the mixed cultures of Azotobacter and Colpoda frequently fixed less nitrogen than the respective pure cultures of Azotobacter. In view of the known oxygen sensitivity of Azotobacter during nitrogen fixation (Parker, 1954, Posgtate, 1971), it was suggested that the ciliates may help to reduce the dissolved oxygen concentrations in the mixed cultures of Azotobacter and Colpoda to more suitable levels for nitrogen fixation between 15 and 25°C. At 28°C the respriatory activity of Azotobacter may be sufficiently large to reduce the dissolved oxygen content to suitable levels in the absence of ciliates. Also, the solubility of oxygen in aqueous media is less at 28°C than at these lower temperatures. This paper reports the results of experiments with shaken and static cultures of Azotobacter chroococcum strain 7115 incubated at 28°C in the presence and absence of Colpoda steini. These experiments were designed as a partial test of the above hypothesis. MATERIALS
AND METHODS
The origin of the microbes and methods of counting the total number of cells were the same as described by Darbyshire (1972). The trophic Colpoda were only counted in cultures that had been incubated for 17, 24 or 31 days, although trophic Colpoda were observed in 371
372
J. F. DARBYSHIRE
the culture flasks on days 3, 10 and 14. The viable numbers of Azotobacter were counted by the drop count method as described by Collins (1964), except that ten drops from each dilution were used. The Azotobacter colonies were counted with an inverted microscope (Zeiss, Oberkiichen) after they had been incubated at 25°C for 2 days. The nutrient agar used (BBJ medium) was the medium described by Brown, Burlingham and Jackson (1962), except that K,HP04 was not sterilized separately. The diluent in the drop count method was BBJ medium without agar or glucose. The liquid medium used in the experiments was the BBJ medium without agar. The cultures were incubated at 28°C either unshaken in an incubator or shaken on an orbital shaker (New Brunswick Scientific Co. Model G.25) at 60 rev/min. The other experimental details were the same as described by Darbyshire (1972). RESULTS
The total quantities of nitrogen and carbon detected in mixed or pure cultures of A chroococcum 7115, when the flasks were incubated unshaken at 28”C, are shown in Table 1. TABLE 1. TOTAL NITROGEN &g/ml) AND TOTAL CARBON (mg/ml) IN PURE A. chroococcum STRAIN 7115 AND MIXED A. chroOCOCCU~STRAIN 7115/C. steini CULTURES INCUBATED AT 28°C and UNSHAKEN
Days after inoculation
A. chroococcum
N
C A. chroococcum and C. steini
N C
14
21
25
28
16 1.5*
37 1.4
43 0.8
51 0.6
11
34 1.3
38 0.9
53 0.5
1.3
* Significantly different (PC 0.05) from the carbon the mixed culture and sampled on the same day.
value in
The static mixed cultures of Azotobacter and Colpoda fixed less nitrogen than the static pure Azotobacter cultures on days 14,21, 25 and 28 (not significant, P
NITROGEN
FIXATION
BY AZOTOBACTER
’ 10 Days
after
1-41:
CHROOCOCCUM
24
373
31
inoculation
FIG. 1. Microbial populations in pure A. chroococcum strain 7115 and mixed A. chroococcunz strain 7115/C. steini cultures incubated unshaken at 28°C. White circles (0) and squares (0) refer to the total and viable Azotobacter populations in pure cultures. Black circles (a), squares (H), diamonds (+) and triangles (N refer to the total and viable Azotobacter populations in mixed CoZpodu/Azorobacter cultures, to the total ciliate populations and to the trophic ciliate populations respectively.
cocci with refractile granules were observed on day 24 in both pure and mixed cultures. At the end of the experiment on day 31, some of the Azotobacter cells in the pure cultures and most Azotobacter cells in the mixed cultures were cysts. A dark pigment was first observed in the majority of the pure cultures on day 21 and in most of the mixed cultures the same pigment appeared on day 22. Subsequently, the pure and mixed cultures were indistinguishable in terms of colour. The chemical analyses (Table 2) from a similar experiment with shaken Azotobacter cultures showed that the shaken mixed cultures fixed an extra 5 pg N/ml more than the shaken pure cultures in the first 2 weeks (not significant, P-C 0 -05). After longer periods of incubation the quantities of nitrogen were almost identical in both pure and mixed shaken cultures. A comparison between all the static and shaken cultures showed that all the static cultures fixed significantly less nitrogen than the shaken cultures in the first 2 weeks (PC O-01) but when the incubation period was for 21 days or longer the nitrogen fixation in the static cultures surpassed significantly the shaken cultures (P < 0.01). The microbial populations of the shaken cultures (Fig. 2) exhibited several differences from that described for static cultures (Fig. 1). The shaken Azotobacter populations in both pure and mixed cultures multiplied more rapidly than the equivalent static cultures in the first 10 days of incubation (significant at PcO.01). The total and viable Azotobacter populations in the shaken pure cultures were significantly larger than the equivalent Azotobacter S.B.B. 413-H
J. F. DARBYSHIRE
374
TABLE 2. TOTAL NITROOEN&g/ml) AND TOTALCARBON(mg/ml) IN PURE A. chroococcum STRAIN 7115 AND MIXED A. chroococcum STRAIN 7115/C. steini CULTURESINCUBATEDAT 28°C AND SHAKENAT 60 rev/min
Days after inoculation
A. chroococarm
A. chroococcum
and C. steini
14
21
25
28
C
18 0.9
25 l.O*
27 0.9
26 0.8
N C
23 0.9
26 0.8
26 0.8
27 0.7
N
* Significantly different (PcO.05) from the carbon value in the mixed culture and sampled on the same day.
FIG. 2. Microbial populations in pure A. chroococcum strain 7115 andmixed A. chroococcum strain 7115/C. steini cultures incubated at 28°C and shaken at 60 rev/min. White circles (0) and squares (0) refer to the total and viable Azotobucter populations in pure cultures. Black circles (O), squares (m), diamonds (+) and triangles (m refer to the total and viable Azotobacter populations in mixed Cofpodu/Azotobucter cultures, to the total ciliate populations and to the trophic ciliate populations respectively.
NITROGEN FIXATION BY AZOTOBACTER
CHROOCOCCUM
375
populations found in shaken mixed cultures sampled on the same day (P < 0 -01) except for total populations on day 4, 17 and 24. These differences in Azatobacter populations between mixed and pure shaken cultures were, however, generally smaller than equivalent differences in Azutobaeter populations described earlier for pure and mixed static cultures. Total ciliate populations in shaken cultures were also much smaller than the total ciliate populations found in static cultures from day 3 onwards (significant at P < O-001). Trophic Colpou’u were observed in the shaken flasks from day 3 onwards and fewer individuals were observed after day 10. The morphological changes in the shaken Azoto~~~te~ populations occurred more rapidly than in the static cultures. Many coccoid rods and some cysts were present in shaken pure and mixed cultures on day 10. The majority of Azotobacter cells in both shaken mixed and pure cultures on day 18 were cocci with refractile granules frequently surrounded by mucilage. Both shaken pure and mixed cultures by day 24 contained large numbers of precystic cocci with refractile granules and cysts. A few of the shaken mixed or pure Azotob~cte$ cultures began to develop a dark pigment on day 4 and most of the shaken flasks had a dark colour by day 7. The shaken pure Azotobacter cultures were slightly darker than shaken mixed cultures of the same age until after day 22 when they were indistinguishable in colour. DI!S&USSiON
A comparison between the chemical analyses for shaken and static cultures of A. chro~COCCU~(Tables 1 and 2) suggest that nitrogen fixation and carbon loss were more rapid in the shaken cultures in the first 2 weeks of incubation. In the third and fourth weeks of incubation there was much less nitrogen fixation in the shaken flasks, especially in the mixed cultures. The microscopical observations of the shaken cultures showed that during the latter 2 weeks there was an increasing proportion of cysts and precystic cocci in the Azotobacter populations and a relative scarcity of actively dividing rods. Jensen (1954) concluded that nitrogen fixation is usually associated with cell proliferation and it would appear that the results from the present shaken experiment support this view. Similarly, the more gradual rate of nitrogen fixation and carbon loss in the static cultures was associated with a slower rate of increase in size of the Azotobacter populations and with the presence of some Azotobacter rods throughout the experiment. If the suggestion that the ciliates are able to maintain Azotobacter cells in a ‘ . . . younger and more active condition. . . ’ is correct (Cutler and Bal 1926), then it might be expected that Azotobacter cysts would be less frequent and rods more frequenr in mixed shaken cultures in the first 2 weeks than in shaken pure Azotobacter cultures of the same age. Although the numbers of Azotobacter cysts were not counted, the shaken mixed cultures did not appear to have a smaller proportion of Azotobacter cysts or a larger proportion of rods in the total bacterial population during the first 2 weeks of incubation compared with shaken pure cultures. The relationship between the presence of trophic Colpoda and smaller populations of Azotobacter in mixed compared with pure cultures previously reported (Darbyshire, 1972), is not so well defined in the present shaken experiment. The small differences between Azotobffcter populations in shaken mixed and pure cultures may be due to the small numbers of trophic Colpodu present in many of the shaken cultures as well as to the more rapid multiplication of Azotobacter in the shaken compared with the static cultures. There was no indication in the shaken or static experiment that ciliate predation affects the viability of the Azotobacter populations. It is still possible, however, that ciliate predation may influence
376
3. F. DARBYSHIRE
the rate of decline of the viable count of Azotobacter in older cultures, which had reachec the so-called growth phase of decline (Cooper, 1971). The much lower total Colpoda populations from day 3 onwards in the shaken compared with the static cultures were associated with the accelerated onset of Azotobacter cyst formation and dark pigmentation in the shaken cultures. Further experiments are in progress to determine whether Azotobacter cysts are intrinsically inedible for Colpoda or whether the dark pigment renders them unsuitable for food. As some of this pigment appears to be released into the mixed culture media, it is possible that such pigments might be released from Azotobacter cells in the soil and discourage Colpoda from devouring adjacent edible food. If this phenomenon has a more general appIjcation to soil protozoa, then such microbial pigments may significantly delay the decomposition of microbial cells in the soil. Any conclusions about the feeding habits of soil protozoa based entirely on monoxenic bacterial/protozoan cultures would also have to be modified. The shaking treatment is Iikely to affect microbial growth and metaboi~sm in several ways besides improving the oxygen transfer rate. Future measurements of the dissolved oxygen concentrations in pure and mixed Azofobacter cultures should provide more direct evidence that protozoa can reduce the dissolved oxygen concentration of Azotobacter cultures significantly. Nevertheless, the results of two experiments described previously (Darbyshire, 1972), showed that Cofpodu steini stimulated nitrogen fixation by Azotobacter to a greater extent when mixed cultures of 100 ml aliquots of unshaken culture fluid in 1 I. Rasks were used, rather than 40 ml aliquots of the same unshaken medium in 150 ml flasks. It is assumed that the oxygen transfer rate in the 1 1. flask experiment was superior to the condition that developed in the smaller flasks. Ack~ow~edgeme~f-the carbon and nitrogen.
author wishes to thank Dr G. ANDERSONwho analysed the culture media for total
REFERENCES BROWN M. E., BURUNGHAM S. K. and JACKSONR. M. (1962) Studies on Azofobacter species in soil---I.
Comparison
of media and techniques for counting Azorobacter in soil. PI. Soil 17, 309-319.
COLLINSC. H. (1964) Microbial Methods, Butterworths, London. COOPERK. E. (1971) Growth of micro-organisms in artificial culture. In Micro-organ&z.r
Fcmction, Form and Environment (L. E. Hawker and A. H. Linton, Eds), pp. 169-192, Arnold, London. CUTLERD. W. and BAL D. V. (1926) In~uence of protozoa on the process of nitrogen fixation by Azofobucfer cbroococcum. Ann. cippl. Biol. 13,516-534. DARBYSHIRE J. F. (1972) Nitrogen fixation by Azofobucfer chroococcum in the presence of Colpodu steini-I. The influence of temperature. Soil Biol. Biochem. 4,359-369. JENSENH. t. (1954) The Azotobacteriaceae. Bucf. Rev. 18, 195-214. PARKERC. A. (1954) Effect of oxygen on the fixation of nitrogen by Azotobacter. Nature, Land. 173,780-781. POSTGATEJ. (1971) Relevant aspects of the physiological chemistry of nitrogen fixation, In ~jcrobe~ and 3iolog~col Prod~cfiv~ty (D. E. Hughes and A. H. Rose, Ed& pp. 287-307, Cambridge University Press.