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Effects of microbial population and culture phosphate composition on the activity of pyrophosphatases from soil microorganisms
EFFECTS OF MICROBIAL POPULATION AND CULTURE PHOSPHATE COMPOSITION ON THE ACTIVITY OF PYROPHOSPHATASES FROM SOIL MICROORGANISMS P. G. E. SEAKLE* and J. D. H~J~HES Deportment of Agriculture, University of Queensland, St. Lucia. JOh7. Queensland, Australia (Acceptrd 7 July 1976) Summary-The quantity of intracellular pyrophosphatases produced in mixed cultures was not related to pyrophosphate or ortho~hosphate concentration in the culture medium, but high pyrffphosp~dte concentration initially depressed microbial growth, delayed the first signs of pyrophosphate hydrolysis and gave rise to a lower initial rate of pyrophosphate hydrolysis in the culture. Not all cultures produced intracellular pyrophosphatases. but when they were present. the same functional kinds of enzymes appeared to be present when compared on the basis of activation by five cations. All cultures inoculated with fresh soil were able to hydrolyse pyrophosphnte in the culture medium, but not all cultures inoculated with stored soil displayed culture pyrophosphatase activity. There was evidence that intracellular pyrophosphatases were responsible for culture pyrophosphatase activity. It was found that cultures showing low intracellular and culture pyrophosphatase activity were dominated by Gram fve rods. When intracellular and culture phosphatase activities were high. Gram -ve rods and Gram -ve cocci dominated the culture populations. As cultures dominated by Gram fve rods were obtained only from stored soil inoculum, it is considered that during soil storage Gram +ve rods became dommant in the soil because of the ability of some members of this group to form resistant endospores.
INTRODUCTION
orthophosphate and providing pyrophosphate as the only source of phosphate. Stored soil was used for inoculation. Details of relevant differences between cultures are given in Table 4. For those cultures to which no orthophosphate was added, potassium and ammonium ions were added as chlorides to compensate for additions normally made as ortllophosph~~t~. A cell free enzyme preparation obtained from each culture was desalted. and the protcin content determined as described previously (Searfe and Hughes, 1977).
In a previous paper (Sea& and Hughes, 1977) the intracellular pyrophosphatases obtained from mixed cultures of soil microorganisms were shown to be activated by a number of cations. No microbial identifi~ation was attempted, but it was concluded that dissimilar populations of microorganisms in various cultures produced only one functional kind of enzyme. This paper examines pyrosphosphate hydrolysis in a number of cultures in relation to their phosphate and microbial composition. using both freshly sampled and stored soil as inocufants. The effect of cufture composition on the pyrophosphatase activity of the extracted enzymes is also investigated. MATERIALS AND
microorganisms were identified during the early fog phase and in the stationary phase by morphology and Gram stain on slides prepared from culture samples. The stained organisms were examined under a microscope using oil immersion and divided into three categories based on the following ratios: Gram fve to Gram -ve rods, Gram +ve to Gram - ve cocci. and rods to cocci. The ratio in each category was estimated to the nearest 25’?< by assigning one of the ratios 0:4, 1:3. 2:2, 3: 1 or 4:0 to it. Five random fields on each slide were examined. Preliminary observations had shown that replicate slides from the same culture gave the same ratios in the 3 categories.
METHODS
Clilruriny
The same basic procedures were adopted as described previously (Searfe and Hughes, f977). Two series of cultures were used. In the first series, six 2 1cultures of soil microorganisms were prepared with compositions as indicated in Table I. Cultures 1 and 2 were inoculated with freshly sampled soil. the others with stored soil. In the second series. two sets of 6 cultures were grown, Set 1 to determine the effect of increasing the concentration of pyrophosphate in the culture, and Set 2 to examine the consequences of withholding * Present address: Department of Agronomy cultural
Science,
llniversity
of Sydney.
Sydney,
RESCLTS AND Intracrllulw
pyrophosphatuw
DISCXSSION
uctivirq
Table 2 gives details of specific activities and specific activity ratios for enzymes extracted from the 6 cultures of Series 1. No activity was obtained for
and HortiAustralia. 157
IFS
P.
G.
E,
SI ~~1.1. md
assays conducted in the alxm~ce of activating cations. Information about the cultttres at time of harvest. including the intracellular p~j-[~ph~sph~lt~~s~activity per litro of culture (C~~~~iJl~~ted from the specific activity for Mg’ + activation) as well as protein harvested per litrc of culture. is prcscntcd in Table 3. Specific activity ratios wcrc similar to those found previously (Scnrlc :mcl Hughes. 1977). Culture 6 sho&s some \xrlation from the others but this is probably dtrc to the fact that for this cirhurc. activity vah~es wcrc small and the error W:IS larpcr. ?lo differences M’LW apparent hct\vccn cnqmcs obtained from cultures using fresh and stored soil as inoculum. Pyrophosphate WIS not noccssary for the production of
J. D.
HUGHI s
pyrophosphatase
(Culture
5). suggesting that the The results indicate that intracellular pyrophosph~~t~~s~s were responsible for culture pyrophosph~~tc hydrolysis. This follows from a comparison of the number of units of enzyme activity per litre of culture (magnesium activation) with culture pyrophosphatase activity. Except for Culture 5, T;tblc 3 shows that the level of culture pyrophosphatnse activity was consistent with the corresponding kvcl of intracellular enzyme activity. but whether enzyme hydrolysis in the culture medium occurs before or after lysis of the microbial cells cannot be positively determined from the rosults. The results also show that not all soil microorganisms produce
enzymes arc constitutivc.
Activil!
MgLA C‘tilturc
MSA*
SAR?
Tahlc 3. ttkct
MS,:“‘-
MSAC‘O’-SAR
SAR
01‘culture treatment
on
culture
MSt?“2’ShR
mcasurc’nlents at harvest
MSAFC2’ShR
(I st series)
Soil pyrophosphatase Table 4. Culture
treatments
Culture
and corresponding
Phosphate Ortho 6mM 6mM 6m.M
Set
6 mM
I
6 mM 6 mM
(i) (ii) (iii)
6mM 6 mM 6mM
(iv) (v) (vi)
nil nil nil
Set 2
specific series)
159
activity activity
ratios
for the extracted
enzymes
Mg
Specific activity Zn co
nil 5mM IOmM 20 mM 40 mM 80 mM Mean
1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.78 0.70 0.86 0.86 0.86 0.86 0.86
0.17 0.13 0. I 6 0. I 6 0. I 5 0.15 0.16
0.13 0.10 0.12 0.13
0.16 0.10 0.15 0.14
0.13
0.15
0.13 0.12
0.16 0. I 5
40 mM 40 mM 40mM Mean 40 mA4 40 mM 40 mM
1.00 1.00 1.00
I.00
0.17 0.17 0.17 0.17 0. I 7 0. I7 0.17 0. I I
0.13 0.13 0.14 0.13 0.13 0.13 0.13
Mean
0.9 I 0.94 0.90 0.92 0.90 0.93 0.90 0.9 1
0.14 0.13 0.12 0.13 0.12 0.12 0.1 I 0.12
treatment Pyro
intracellular pyrophosphatases, since none were obtained from Cultures 3 and 4 (Table 3). The pH-activity profiles associated with the Series 2 cultures were the same as those obtained by Searle and Hughes (1977) for each of the activating cations. The similarity of the calculated specific activity ratios (Table 4) indicates that variations in the amounts of orthophosphate and pyrophosphate in the culture medium had no effect on the functional kind of enzyme produced by the microorganisms. Neither of the phosphate species was shown to be essential for the production of active intracellular pyrophosphatases, and there was no evidence that pyrophosphate affected the quantity of enzyme produced. There was, for example, no definite relationship between intracellular pyrophosphatase activity per unit volume of culture and pyrophosphate concentration in the culture. This result discounts the idea that higher concentrations of pyrophosphate favour microorganisms containing pyrophosphatases, or that pyrophosphate induces pyrophosphatase production. For the orthophosphate treated cultures. there was no significant difference in activity per unit volume of culture between the two concentrations of orthophosphate. This evidence suggests that the pyrophosphatases are constitutive.
The pattern of culture pyrophosphatase activity with time depended on the concentration of pyrophosphate in the culture. Where the added pyrophosphate was least (5 mM). the rate of hydrolysis was initially high. but decreased almost to zero. This was in contrast to cultures with higher additions of pyrophosphate, which exhibited an initially low rate, increasing with time to a maximum at or shortly before the stationary phase. The decrease in hydrolysis rate with time for the low pyrophosphate culture was due to the fact that all the pyrophosphate was quickly hydrolysed. The trend for initiation of culture pyrophosphatase activity to be delayed by high concentrations of pyro-
1.00 1.00 1.00 I.00
ratio Mn
(2nd
0.13
Fe
phosphate was probably due to the suppression of growth by pyrophosphate during most of the log phase. This is in agreement with the work of Hashimoto et ul. (1969) who found that increasing concentrations of ammonium pyrophosphate inhibited the growth of soil microorganisms and reduced pyrophosphate hydrolysis. At the stationary phase the rate of hydrolysis was not related to initial pyrophosphate concentration, suggesting that adjustments in the microbial population had occurred enabling pyrophosphatase activity to rise. The effect of orthophosphate concentration in the culture was minimal, but there was a tendency during the early stages for hydrolysis to be more rapid in the nil-orthophosphate cultures. despite the fact that there were no corresponding differences in culture growth. This trend did not persist beyond about 30 h, after which the rate of hydrolysis increased dramatically in all cultures. This suggests that despite the initial effect, microbial population re-adjustments were able to compensate before the onset of the stationary phase. Microhid
populafiorl
The observations made on the cultures and recorded in Table 3 suggest differences in the composition of the microbial populations. This is confirmed in Table 5 which shows that at two sampling times, the compositions of the population varied widely from one culture to another. Even the first 2 cultures. which are duplicates of the same treatment, show differences at each sampling time. Between the 2 sampling times all cultures showed some variation in the composition of the microbial population, which indicates interactions between the microorganisms present in the various cultures. A comparison of pyrophosphatase activity and microbial composition reveals that cultures lacking or low in culture pyrophosphatase or intracellular pyrophosphatase activity were dominated by Gram +ve rods. This is obvious by comparing culture pyrophos-
P. G. E. SEAKLI: and J. D. HI GHI;S
160 Table 5. Composition
of culture
populations of soil microorganisms based on morphology stain at two stages of growth (1st series) Growth
Culture* Composition
Culture
*Gram
+ve:
Gram
and Gram
stage
karly
log phase
Stationary
phase
I :3
I :3
I:3
I:3
I :3
0:4
0:4
0:4
0:4
0:4
0 :4
0 :4
0:4
0:4
0 :4
0: 4
0:4
0: 4
O:4
0:4
7.1 -.-
7.-l _.A
7.7 _.I
7.7 -.-
4:o
4:O
4:0
3:O
4:o
7.7 _.-
?:I
3:i
?:I!
3:l
3:i
7.7 _.I
7.7 _.-
2:’
2:?.
2:2
0:J
0:4
0:4
0 :4
0:4
I:3
0:4
0:4
0:4
014
3.7 A...
2:2
4:0
4:0
4:O
4:0
4 :0
2:2
212
7.3 -.-
4:o
4:O
4:O
4:0
4:o
4:o
4:0
4:o
4:0
4:o
c:c
O:4
if:4
O:J
0:-I
0:4
O:3
0:4
0: 4
0:4
0:4
R:C
3:l
?:I
‘12
3:l
3:l
4:o
4:o
4:o
4:O
4:o
R:R
4:0
4:O
4 :0
4:o
4:o
4:o
4:0
4:o
4:O
4:o
i.:c
0:4
0:4
0:3
0:4
0:4
4:o
4: 0
4:o
4:o
4:o
R:C
4:0
2:2
2:2
.i:I
3:1
i:l
4:o
4:o
4:0
4:o
ii:?
3:l
212
7.3 _._
3:l
3:1
?.) . . ..-
I:3
I:3
1.7 _.._
2:2
t :C‘
0 :4
0 :4
0:J
0:4
0:4
0:4
O:4
0: 4
O:4
0:4
R:C
3:l
3:l
3:l
3:l
3:l
3:2
2:7
7.3 _._
2:2
2:2
R : Ii
4:O
4:O
4:O
4:0
4:o
4:o
4:0
410
4: 0
4:o
(.:c_
0:4
0:4
0:4
0:4
ii:4
0:4
I:3
0: 4
0:J
1:3
R:C
4:0
4:O
4:O
4:0
4:o
4:o
4:0
4:O
4:o
4:O
-vc
rods;
Gram
+ve:
Gram
phatase activity and intracellular pyrophosphatase activity units per litre (Mg’+ activation) for Cultures 3.4 and 6 with their microbial compositions. Whether the Gram +ve rods belong to the same species is not known and it cannot be generalised that other Gram + ve rods from soils do not contain intracellular pyrophosphatases. Blumenthal et NI. (1967) for example, have shown that all the Gram +ve rods which they examined contained intracellular pyrophosphatases, so it is likely that some Gram +ve rods in soils do contain thcsc enzymes. intracellular pyrophosphatase activity was probably present in the Gram -ve rods and/or Gram -ve cocci. e.g. in Cultures I. 2 and 5. whose culture and;or intracellular pyrophosphatase activities were high. However. this assumes that the Gram +ve rods did not contribute to activity in Cultures 2 and 5 on the basis of the evidence above. Activity may be present in both Gram - vc rods and Gram - vc cocci because Blumenthal et LII.(1967) have found intraccllular pyrophosphatases in all examinations of organisms of these two forms. Soit storage may have caused loss of pyrophosphatase activity due to a relative increase in the proportion of Gram +ve rods in the microbial population.
-vc
cocci:
rods: cocci
Results in Tables 2 and 3 indicate that loss of activity occurred only where the populations were predominantly Gram +ve rods (Cultures 3. 4 and 6). This suggests that the loss in enzyme activity with time is due to changes in the microbial population rather than a loss in the ability of the same microorganisms to produce the enzymes. This indication that storing soil leads to an increased proportion of Gram +ve rods is consistent with known characteristics of some members of this group of organisms as being able to form resistant endospores and so endure longer than non spore-formers.
REFERENCES B. I., JOHNSON M. K. and JOHNSOU E. J. (1967) Distribution of heat-labile and heat-stable inorganic pqrophosphatases among some bacteria. Cil&. d. Microhiol. 13. 1695-l 699. HASHIMOW I.. HI;GHF:S J. D. and PHII.I,U 0. D. (1969) Reactions of triammonium pyrophosphnte with soils and soil minerals. Soil Set. .%c. A0z. Proc. 33. 401~405. SEAKLE P. G. E. and HWHFS J. D. (1977) T1v.z activity of pyrophospll~lt~lses from soil microorganisms: I. Cation activation. Sotl Bioi. Bio&t~~ 9. 153-l S6.
RLLMENTHAL
INTRODUCTION
orthophosphate and providing pyrophosphate as the only source of phosphate. Stored soil was used for inoculation. Details of relevant differences between cultures are given in Table 4. For those cultures to which no orthophosphate was added, potassium and ammonium ions were added as chlorides to compensate for additions normally made as ortllophosph~~t~. A cell free enzyme preparation obtained from each culture was desalted. and the protcin content determined as described previously (Searfe and Hughes, 1977).
In a previous paper (Sea& and Hughes, 1977) the intracellular pyrophosphatases obtained from mixed cultures of soil microorganisms were shown to be activated by a number of cations. No microbial identifi~ation was attempted, but it was concluded that dissimilar populations of microorganisms in various cultures produced only one functional kind of enzyme. This paper examines pyrosphosphate hydrolysis in a number of cultures in relation to their phosphate and microbial composition. using both freshly sampled and stored soil as inocufants. The effect of cufture composition on the pyrophosphatase activity of the extracted enzymes is also investigated. MATERIALS AND
microorganisms were identified during the early fog phase and in the stationary phase by morphology and Gram stain on slides prepared from culture samples. The stained organisms were examined under a microscope using oil immersion and divided into three categories based on the following ratios: Gram fve to Gram -ve rods, Gram +ve to Gram - ve cocci. and rods to cocci. The ratio in each category was estimated to the nearest 25’?< by assigning one of the ratios 0:4, 1:3. 2:2, 3: 1 or 4:0 to it. Five random fields on each slide were examined. Preliminary observations had shown that replicate slides from the same culture gave the same ratios in the 3 categories.
METHODS
Clilruriny
The same basic procedures were adopted as described previously (Searfe and Hughes, f977). Two series of cultures were used. In the first series, six 2 1cultures of soil microorganisms were prepared with compositions as indicated in Table I. Cultures 1 and 2 were inoculated with freshly sampled soil. the others with stored soil. In the second series. two sets of 6 cultures were grown, Set 1 to determine the effect of increasing the concentration of pyrophosphate in the culture, and Set 2 to examine the consequences of withholding * Present address: Department of Agronomy cultural
Science,
llniversity
of Sydney.
Sydney,
RESCLTS AND Intracrllulw
pyrophosphatuw
DISCXSSION
uctivirq
Table 2 gives details of specific activities and specific activity ratios for enzymes extracted from the 6 cultures of Series 1. No activity was obtained for
and HortiAustralia. 157
IFS
P.
G.
E,
SI ~~1.1. md
assays conducted in the alxm~ce of activating cations. Information about the cultttres at time of harvest. including the intracellular p~j-[~ph~sph~lt~~s~activity per litro of culture (C~~~~iJl~~ted from the specific activity for Mg’ + activation) as well as protein harvested per litrc of culture. is prcscntcd in Table 3. Specific activity ratios wcrc similar to those found previously (Scnrlc :mcl Hughes. 1977). Culture 6 sho&s some \xrlation from the others but this is probably dtrc to the fact that for this cirhurc. activity vah~es wcrc small and the error W:IS larpcr. ?lo differences M’LW apparent hct\vccn cnqmcs obtained from cultures using fresh and stored soil as inoculum. Pyrophosphate WIS not noccssary for the production of
J. D.
HUGHI s
pyrophosphatase
(Culture
5). suggesting that the The results indicate that intracellular pyrophosph~~t~~s~s were responsible for culture pyrophosph~~tc hydrolysis. This follows from a comparison of the number of units of enzyme activity per litre of culture (magnesium activation) with culture pyrophosphatase activity. Except for Culture 5, T;tblc 3 shows that the level of culture pyrophosphatnse activity was consistent with the corresponding kvcl of intracellular enzyme activity. but whether enzyme hydrolysis in the culture medium occurs before or after lysis of the microbial cells cannot be positively determined from the rosults. The results also show that not all soil microorganisms produce
enzymes arc constitutivc.
Activil!
MgLA C‘tilturc
MSA*
SAR?
Tahlc 3. ttkct
MS,:“‘-
MSAC‘O’-SAR
SAR
01‘culture treatment
on
culture
MSt?“2’ShR
mcasurc’nlents at harvest
MSAFC2’ShR
(I st series)
Soil pyrophosphatase Table 4. Culture
treatments
Culture
and corresponding
Phosphate Ortho 6mM 6mM 6m.M
Set
6 mM
I
6 mM 6 mM
(i) (ii) (iii)
6mM 6 mM 6mM
(iv) (v) (vi)
nil nil nil
Set 2
specific series)
159
activity activity
ratios
for the extracted
enzymes
Mg
Specific activity Zn co
nil 5mM IOmM 20 mM 40 mM 80 mM Mean
1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.78 0.70 0.86 0.86 0.86 0.86 0.86
0.17 0.13 0. I 6 0. I 6 0. I 5 0.15 0.16
0.13 0.10 0.12 0.13
0.16 0.10 0.15 0.14
0.13
0.15
0.13 0.12
0.16 0. I 5
40 mM 40 mM 40mM Mean 40 mA4 40 mM 40 mM
1.00 1.00 1.00
I.00
0.17 0.17 0.17 0.17 0. I 7 0. I7 0.17 0. I I
0.13 0.13 0.14 0.13 0.13 0.13 0.13
Mean
0.9 I 0.94 0.90 0.92 0.90 0.93 0.90 0.9 1
0.14 0.13 0.12 0.13 0.12 0.12 0.1 I 0.12
treatment Pyro
intracellular pyrophosphatases, since none were obtained from Cultures 3 and 4 (Table 3). The pH-activity profiles associated with the Series 2 cultures were the same as those obtained by Searle and Hughes (1977) for each of the activating cations. The similarity of the calculated specific activity ratios (Table 4) indicates that variations in the amounts of orthophosphate and pyrophosphate in the culture medium had no effect on the functional kind of enzyme produced by the microorganisms. Neither of the phosphate species was shown to be essential for the production of active intracellular pyrophosphatases, and there was no evidence that pyrophosphate affected the quantity of enzyme produced. There was, for example, no definite relationship between intracellular pyrophosphatase activity per unit volume of culture and pyrophosphate concentration in the culture. This result discounts the idea that higher concentrations of pyrophosphate favour microorganisms containing pyrophosphatases, or that pyrophosphate induces pyrophosphatase production. For the orthophosphate treated cultures. there was no significant difference in activity per unit volume of culture between the two concentrations of orthophosphate. This evidence suggests that the pyrophosphatases are constitutive.
The pattern of culture pyrophosphatase activity with time depended on the concentration of pyrophosphate in the culture. Where the added pyrophosphate was least (5 mM). the rate of hydrolysis was initially high. but decreased almost to zero. This was in contrast to cultures with higher additions of pyrophosphate, which exhibited an initially low rate, increasing with time to a maximum at or shortly before the stationary phase. The decrease in hydrolysis rate with time for the low pyrophosphate culture was due to the fact that all the pyrophosphate was quickly hydrolysed. The trend for initiation of culture pyrophosphatase activity to be delayed by high concentrations of pyro-
1.00 1.00 1.00 I.00
ratio Mn
(2nd
0.13
Fe
phosphate was probably due to the suppression of growth by pyrophosphate during most of the log phase. This is in agreement with the work of Hashimoto et ul. (1969) who found that increasing concentrations of ammonium pyrophosphate inhibited the growth of soil microorganisms and reduced pyrophosphate hydrolysis. At the stationary phase the rate of hydrolysis was not related to initial pyrophosphate concentration, suggesting that adjustments in the microbial population had occurred enabling pyrophosphatase activity to rise. The effect of orthophosphate concentration in the culture was minimal, but there was a tendency during the early stages for hydrolysis to be more rapid in the nil-orthophosphate cultures. despite the fact that there were no corresponding differences in culture growth. This trend did not persist beyond about 30 h, after which the rate of hydrolysis increased dramatically in all cultures. This suggests that despite the initial effect, microbial population re-adjustments were able to compensate before the onset of the stationary phase. Microhid
populafiorl
The observations made on the cultures and recorded in Table 3 suggest differences in the composition of the microbial populations. This is confirmed in Table 5 which shows that at two sampling times, the compositions of the population varied widely from one culture to another. Even the first 2 cultures. which are duplicates of the same treatment, show differences at each sampling time. Between the 2 sampling times all cultures showed some variation in the composition of the microbial population, which indicates interactions between the microorganisms present in the various cultures. A comparison of pyrophosphatase activity and microbial composition reveals that cultures lacking or low in culture pyrophosphatase or intracellular pyrophosphatase activity were dominated by Gram +ve rods. This is obvious by comparing culture pyrophos-
P. G. E. SEAKLI: and J. D. HI GHI;S
160 Table 5. Composition
of culture
populations of soil microorganisms based on morphology stain at two stages of growth (1st series) Growth
Culture* Composition
Culture
*Gram
+ve:
Gram
and Gram
stage
karly
log phase
Stationary
phase
I :3
I :3
I:3
I:3
I :3
0:4
0:4
0:4
0:4
0:4
0 :4
0 :4
0:4
0:4
0 :4
0: 4
0:4
0: 4
O:4
0:4
7.1 -.-
7.-l _.A
7.7 _.I
7.7 -.-
4:o
4:O
4:0
3:O
4:o
7.7 _.-
?:I
3:i
?:I!
3:l
3:i
7.7 _.I
7.7 _.-
2:’
2:?.
2:2
0:J
0:4
0:4
0 :4
0:4
I:3
0:4
0:4
0:4
014
3.7 A...
2:2
4:0
4:0
4:O
4:0
4 :0
2:2
212
7.3 -.-
4:o
4:O
4:O
4:0
4:o
4:o
4:0
4:o
4:0
4:o
c:c
O:4
if:4
O:J
0:-I
0:4
O:3
0:4
0: 4
0:4
0:4
R:C
3:l
?:I
‘12
3:l
3:l
4:o
4:o
4:o
4:O
4:o
R:R
4:0
4:O
4 :0
4:o
4:o
4:o
4:0
4:o
4:O
4:o
i.:c
0:4
0:4
0:3
0:4
0:4
4:o
4: 0
4:o
4:o
4:o
R:C
4:0
2:2
2:2
.i:I
3:1
i:l
4:o
4:o
4:0
4:o
ii:?
3:l
212
7.3 _._
3:l
3:1
?.) . . ..-
I:3
I:3
1.7 _.._
2:2
t :C‘
0 :4
0 :4
0:J
0:4
0:4
0:4
O:4
0: 4
O:4
0:4
R:C
3:l
3:l
3:l
3:l
3:l
3:2
2:7
7.3 _._
2:2
2:2
R : Ii
4:O
4:O
4:O
4:0
4:o
4:o
4:0
410
4: 0
4:o
(.:c_
0:4
0:4
0:4
0:4
ii:4
0:4
I:3
0: 4
0:J
1:3
R:C
4:0
4:O
4:O
4:0
4:o
4:o
4:0
4:O
4:o
4:O
-vc
rods;
Gram
+ve:
Gram
phatase activity and intracellular pyrophosphatase activity units per litre (Mg’+ activation) for Cultures 3.4 and 6 with their microbial compositions. Whether the Gram +ve rods belong to the same species is not known and it cannot be generalised that other Gram + ve rods from soils do not contain intracellular pyrophosphatases. Blumenthal et NI. (1967) for example, have shown that all the Gram +ve rods which they examined contained intracellular pyrophosphatases, so it is likely that some Gram +ve rods in soils do contain thcsc enzymes. intracellular pyrophosphatase activity was probably present in the Gram -ve rods and/or Gram -ve cocci. e.g. in Cultures I. 2 and 5. whose culture and;or intracellular pyrophosphatase activities were high. However. this assumes that the Gram +ve rods did not contribute to activity in Cultures 2 and 5 on the basis of the evidence above. Activity may be present in both Gram - vc rods and Gram - vc cocci because Blumenthal et LII.(1967) have found intraccllular pyrophosphatases in all examinations of organisms of these two forms. Soit storage may have caused loss of pyrophosphatase activity due to a relative increase in the proportion of Gram +ve rods in the microbial population.
-vc
cocci:
rods: cocci
Results in Tables 2 and 3 indicate that loss of activity occurred only where the populations were predominantly Gram +ve rods (Cultures 3. 4 and 6). This suggests that the loss in enzyme activity with time is due to changes in the microbial population rather than a loss in the ability of the same microorganisms to produce the enzymes. This indication that storing soil leads to an increased proportion of Gram +ve rods is consistent with known characteristics of some members of this group of organisms as being able to form resistant endospores and so endure longer than non spore-formers.
REFERENCES B. I., JOHNSON M. K. and JOHNSOU E. J. (1967) Distribution of heat-labile and heat-stable inorganic pqrophosphatases among some bacteria. Cil&. d. Microhiol. 13. 1695-l 699. HASHIMOW I.. HI;GHF:S J. D. and PHII.I,U 0. D. (1969) Reactions of triammonium pyrophosphnte with soils and soil minerals. Soil Set. .%c. A0z. Proc. 33. 401~405. SEAKLE P. G. E. and HWHFS J. D. (1977) T1v.z activity of pyrophospll~lt~lses from soil microorganisms: I. Cation activation. Sotl Bioi. Bio&t~~ 9. 153-l S6.
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