DETECTION OF SrDEROP~ORES IN SOIL BY A ... - Science Direct

SoilBkd.&drem. Vol.18,No. 5,pp.481-486,1986 Printed in Great Britain

0038-0717/86 %3.00+0.00 Pergamon Journals Ltd

DETECTION OF SrDEROP~ORES IN SOIL BY A DIRECT BIOASSAY P. BOSSIER and W. VERSTRAETE University of Gent, Faculty of Agricultural Sciences, Laboratory for Microbial Ecology, Coupure L 653, 9000 Gent, Beigium (Accepted 28 February 1986) Summary-A bioassay has been developed for the detection of siderophores in soil. The siderophore concentration can be determined directly, avoiding extraction. The growth of the siderophore-auxotrophic bacterium ArzhrobacterJG-9 in soil suspension cultures is used as a measure of the amount of siderophores present in the suspended soil. To measure growth, the amount of organic carbon metabolized is determined. Growth in the soil suspension cultures was governed by both the siderophore ~n~nt~tion and the iron availability. Since both factors are soil dependent, the bioassay has been based on the principle of standard addition. Ferrioxamine B is used as internal standard. The lower threshold of siderophores in soils, detectable by the assay, is of the order of 5 /Ig ferrioxamine B equivalent activity kg-’ soil. The variability coefficient of the assay is of the order of 11%.

~RODU~ION

Siderophores are extracellular microbial compounds with a very high affinity for Fe3+-ions (Neilands, 1984). The ecological significance of these compounds is the subject of increasing scientific interest. In order to study the occurrence and importance of these compounds in the soil ecosystem, several bioassays have already been developed (Akers, 1981, 1983; Powell et uZ., 1980, 1983). They all have the common feature that they determine the siderophore concentration in aqueous soil extracts. However, the extraction of siderophores from the soil is incomplete and undefined. For instance, when ferrioxamine B is supplied to soils (1 pg g-’ air-dry soil) on the average only 3.7% with a variation from 0.3 to 17.3% can be recovered (Powell et al., 1980). As this is a serious draw-back to research on the ecological significance of siderophores we have designed a new bioassay, allowing direct contact between the siderophores present in a soil sample and the bioassay microorganism. MATERIALS AND METHODS

Bioassay

In all experiments the natural siderophoreauxotrophic bacterium, Arthrobacter JG-9 was used as a bioassay ~croorganism. This strain has been reported to be stimulated by most fungal hydroxamate-siderophores (Bumham and Neilands, 1961; Frederick et al., 1981) and does not respond to other types of iron-chelators, such as 2,3 dihydroxy benzoic acid and enterochelin (Powell et al., 1980). It was not found to react to fluorescent siderophores produced by ~se~~o~o~ spp (unpublish~ results). The ArFhrobacFer JG-9 strain was obtained from the laboratory of J. Szaniszlo, Department of Microbiology, University of Texas at Austin (U.S.A.). Ferrioxamine B, commercially available as Desferal@, was purchased from Ciba-Geigy. This siderophore was used as an internal standard in the

bioassay and as a reference siderophore in other experiments. This compound is heat stable. The bioassay medium was composed of (pi-‘): sucrose 4.55; casamino acids 1.05; yeast extract 1.05; MgS0,*7H,O 0.05; NH&l 0.11; Tris 2.50 and CaCO,2.00. The pH of the medium was adjusted with 10% HCl to 7.9 before the addition of CaCO,. Bioassay cultures (2OOml medium in 5OOml Erlenmeyers) were incubated on a rotary shaker (150 rev min-‘) at 28°C for 72 h. The bioassay cultures were inoculated with 1 ml of a culture containing 3.75 pg ferrioxamine Bl-’ and grown under the abovementioned conditions. No growth was detected in blank cultures in which ferrioxamine 3 carried over with the inoculum was the sole source of siderophores. Growth in the bioassay cultures was delined as the difference in total organic carbon content (TOC, mgC I-‘) of the medium after sterilisation and after 72 h of growth. To determine this organic C consumption, samples were taken aseptically and centrifuged for 10 min at 20,OOOg. TGC was measured in the supematant. The TOCequipment, with a measuring range between 0 and 1000 mg C l-l, was purchased from Maihak (Hamburg, Germany). All bioassay cultures were duplicated. The organic C-consumption values presented are the means of the organic Cconsumption in the two cultures. Soils

Some characteristics of the soils used in the experiments are summarized in Table 1. Fulvic and humic acids were extracted from soil according to the method described by Schnitzer (1982). Humic acids were not further purified and consequently consisted of 70% ash (mainly NaCl). Behaviour of ferrioxamine

B in soil suspensions While ferrioxamine B upon dissolution in water yields a colourless solution, the ferri-form shows a maximum absorption at 430 nm. This characteristic

482

P. BOSIER and W. VERSTRAETE Table 1. Some properties of the soils used in the bioassays

Soil B G Ml M3 M4

Texture Sand Sandy Sandy Sandy Sandy

Land use Grassland Grassland Grassland A-Gr= Arable

clay loam loam loam

PH KG

%C

EDTA-Fe (mg Fe kg-’ soil)

6.3 6.5 6.0 6.1 6.1

3.1 4.0 1.8 1.2 0.9

ND 446 422 298 177

Siderophore FOB-value (pg kg-’ soil) 70 31 72 60 24

‘Triennial rotation grassland-arable. ND = Not determined.

was used to determine the iron-mobilizing capacity and the behaviour of the siderophore in a soil suspension (Fig. 1). A ferrioxamine B solution (380 FM) was buffered with 15 g Tris 1-l. The pH was adjusted to 7.9. In order to saturate the iron-chelator 380 PM Fe3+ (as FeCi,*6H,O) was added. This solution had an absorbance (430 nm) of 1.008. In 100 ml containing either the ferri-form (a) or the defer&form (B) of ferrioxamine B, 10 g of soil was added and shaken for 30 min (150 rev min-‘). The soil was separated from the liquid by centrifugation (10 min, 20,OOOg). The supernatant was subsequently filtered (0.45 pm). In theory, the ferrioxamine B can become partitioned between the soil and the solution, both in the deferri and in the ferri-form. This $elds four fractions: adsorbed fe~fe~oxamine B and defe~fe~oxamine B and dissolved fe~fer~oxamine B and def~fer~ox~ine B. The absorbance of the ferriferrioxamine B solution (ar) after contact with a soil yields the partition coefficient (PC) of the ironsaturated siderophore between the solution and the

d

/

Ferrioxamina6 (250 mg I-’ or 380 FM)

Iron form (tbs.*: 1.008) (21.26 mg Fe I;’ 01 380 8~) lOOml++lOg

soil-humus complex. After 30 min of contact of defer~fer~ox~ine B (8) with log of soil, the siderophore will have mobilized some Fe3+ from the soil-humus complex which can be measured by absorbance (x2). After saturation of an aliquot of the supematant with Fe3+, the absorbance increases (x3). The part of the Fe’+ that does not get chelated precipitates, due to the high pH, in flakes which can be separated from the liquid by centrifugation. The value xj represents the dissolved ferrioxamine B which was present either in the ferri or defer+form. The difference between 1.008 and x3 yields the adsorbed ferrioxamine B, both in the ferri and in the deferri form. The difference between x, and x, gives the dissolved defer~fer~oxamine B Cy2). Since part of the formed fe~fe~oxamine B becomes adsorbed to the soil-humus complex, as represented by the partition coefficient (PC), the total amount of the ferriferrioxamine B formed during the 30min of contact can be calculated by dividing x, by PC. The difference between that value and the

\

Non-iron form 1 1OOm1+10g

soil

soil

2 Shaking 30 min 150 rev min-’

1 Shaking 30 min 160 rev min-’

1 Centrifuge, filter (0.45 pm)

1 Centrifuge, filter (0.45 pm) J \ Measure abs.: x2 add to 50 ml aliquot 1.06 mg Fe

J Measure abs.: x, Xl -3

1

1 Allow to equilibrate overmght and centrifuge

p.c.t

1

1.008

Measure abs.: x3

‘abs.: absorbance tP.C.: partition coefficient of ferriferrioxamine 8.

Calcutation 5: dissolved (ferriferrioxamine6 end deferriferrioxamine B): 1.008 - x,: adsorbed (ferrif~rioxamine 6 and deferriferrioxamine 6); x2: dissolved ferrif~rioxamine B ( y, ): x,-x2: dissolved deferrife~ioxamine B ( y2): 2. dissolved and adsorbed ferriferrioxamine B: P.C.‘ &-x2: (l.OOB-X,)-(&-X,): by dividing y,, percentages.

y2,ys and

adsorbed ferriferrioxamine B ( y3); adsorbed deferriferrioxamine B ( yr);

y1 by 1.008 and multiplying by 100, the four fractions are expressed in

Fig. 1. Procedures for the examination of the behaviour of ferrioxamine B in soil suspension.

Bioassay for siderophores

L

I-

v

o

483

I

2000

.E .s

1500

z E 2

1000

z

j

I/.-’

‘-y , , , , , 0

10

20

30

Ferrioxomine

40

B (pg

50

I-‘)

Ferrioxomine

B

(pg

I-‘)

Fig. 2. Relationship between ferrioxamine B concentration and the growth of Arthrobacrer JG-9 as measured by organic C-consumption (TOC, mg C 1-r).

Fig. 4. Growth of Arthrobacter JG-9 in stimulatory soil suspension cultures. + __ + with 1 g soil (soil G); iJ----0 without soil.

B (x1) yields the adsorbed The adsorbed deferriferrioxamine B (yd) is obtained by the difference between the three known fractions (y,, y, and ys) and the value 1.008 (absorbance of the 380 PM ferrioxamine B solution completely saturated with Fe). For convenience, the four fractions are expressed as percentages.

which y stands for the organic C-consumption (mg C l-i), x for the siderophore concentration, and a and b being two constants specific for the soil examined. In the presence of a small amount of soil, added to the cultures before autoclaving, similar dose response curves could be obtained (Figs 4 and 5). However some soils appeared to stimulate the growth of Arthrobacter JG-9 (Fig. 4), while other soils had an inhibitory effect (Fig. 5). Based on this model, the bioassay was designed as shown in Table 2. By substitution of a and b, the unknown amount of siderophore present in the suspended soil x can be calculated. The value x is expressed in pg kg-’ soil and represents in fact a ferrioxamine B equivalent activity (abbreviated as FOB-value) towards Arthrobacter JG-9. The amount of soil used in the bioassay had to be adjusted for each sample in order to obtain a detectable organic C-consumption in the culture where soil is the sole source of siderophore (culture I). An experiment was set up to evaluate the possible interference of the amount of soil used in the bioassay, on the calculated FOB-value. The results summarized in Table 3 show that the growth of

dissolved ferriferrioxamine ferriferrioxamine B &).

RESULTS

The siderophore-auxotrophic bacterium Arthrobatter JG-9 can use a variety of hydroxamate type of iron chelators as a growth factor. The relationship between the ferrioxamine B concentration and the growth of the bioassay microorganism, under the described experimental conditions, is given in Fig. 2. A linear (P = 0.01) regression could be drawn between the reciprocal values of the organic C consumption, as measurement of growth, and the reciprocal values of the ferrioxamine B concentration (Fig. 3). Consequently the relationship between the siderophore concentration and the growth of Arthrobacter JG-9 can be described by the model y = ax/(b +x), in

E10

,j 0.2

0.4

Ferrioxomine

0.6 B (pg

0.8

1.0

I-‘)-’

“““f/, , , , 0

10

20 Ferrioxomine

Fig. 3. Linear regression between the reciprocal values of ferrioxamine B concentration and organic C consumption as measure of growth (TOC, mg Cl-‘) for cultures of Arlhrobucrer JG-9 (r = 0.997, P = 0.01, n = 12).

30 B (pg

40

( 50

I-‘)

Fig. 5. Growth of Arthrobacter JG-9 in inhibitory soil suspension cultures. +-+ with 2g soil (soil M3); O-0 without soil.

P. BOSSIER and

484

W. VERSTRAETE

Table 2. Design of the bioassay Culture I Siderophore source

Equation

z g soil

&x8%

Culture II

Culture III

z g soil + 0.5 fig fenioxamine

z g soil + 2 pug ferrioxamine B

B

a(x + 2)

, _ 0

+ 0.5) b +x + 0.5

“=m

k,l,m: organic C-consumption in the respective cultures. x: amount of siderophore present in r g soil. a,b: two unknown constants (soil dependent). x is calculated by substitution of D and b. k, I and m are the means of two replicates, in total 6 cuhures determine x.

are used to

Table 3. Behaviour of the bioassay as influenced by using different amounts of soil (soil B) Siderophore Culture

SOUrCe

Organic C consumption (mgCI-‘)

FOB-value olg per 290 mI)

FOB-value &,g kg-’ soil)

Series A I II III

2.50 g soil 2.50 g soil + 0.5 pg ferrioxamine B 2.50 g soil + 2 pg ferrioxamine B

155 565 1345

0.157

63

Series B I II III

1.25g soil 1.25 g soil + 0.5 cg ferrioxamine B I .25 g soil + 2 pg ferrioxamine B

85 495 1305

0.089

72

Arthrobacter JG-9 is better in the bioassay cultures where more soil is used. The calculated FOB-values, expressed in pg kg-’ soil, are not significantly different as tested by a t-test. Consequently the bioassay was considered to behave,S for the given boundary conditions, independently from the amount of soil suspended. The accuracy of the bioassay was examined by performing the bioassay on four replicates from the same soil sample (Table 4). The FOB-value for that soil was on the average 36 pg kg-’ soil with a standard deviation of 4.2. This corresponds to a coefficient of variation of 11.8%. In the bioassay for vitamin B,, with Lactobacilks leichmanii a similar coefficient of variation has been obtained (Hansen and Hauschildt, 1974). A special test was set up to verify the recovery of ferrioxamine B from soil. Two soils with a different behaviour in the bioassay i.e. a stimulatory soil (soil M 1) and an inhibitory soil (soil M4) were used in this experiment. By means of an atomizer, 2OOpg ferrioxamine B was equally distributed within 1 kg soil. In the bioassay cultures in which both blank soils (no ferrioxamine B added to the soil) served as sole siderophore source (cultures I) no detectable organic C cons~ption was observed (Table 5). Consequently, the calculated FOB-value is zero. The growth response in the cultures I, in which ferrioxamine B supplemented soil samples were suspended, is detectable and different for both soils. However, irrespective of the behaviour of the bioassay microorganism in these soil suspension cultures, the suppl~ented fer~oxa~ne B could be totally recovered. The calculated FOB-values for both ferrioxamine B-amended soil samples were very close to the assumed value of 200 pg kg-’ soil indicating that the design of the bioassay permits quantitative recovery. The FOB-values of soil Ml and M4 (blank soils, Table 5) are different to those given in

Table 1. In Table 1 the FOB-values are fresh field samples, while the data in Table 5 were obtained after the same soil samples had been stored wet for several months. Apparently siderophores present in the samples become catabolized during the prolonged storage of the moist samples. The experiment summarized in Table 5, indicates that the growth potential of Arthrobacter JG-9 in soil suspension cultures does not influence the determination of the siderophore concentration in a soil sample. Yet, this difference in growth response of Atthrobacter JG-9 to equal amounts of growth factor in soil suspension cultures evokes questions about the nature of that inhibition or stimulation. Adsorption of the siderophore was postulated to be the underlying mechanism. When fulvic and humic acids were added to cultures of Arthrobacter JG-9 containing 0.5 pg ferrioxamine B per 206 ml, a very significant reduction of growth was noticed (Table 6). Salt present with the unpurified humic acid, could not have caused such a strong inhibition. Highly purified sand, with a very low adsorption capacity, gave only a small inhibition, indicating that physical disturbance of Arthrobacter JG-9 by the presence of ‘soil is of minor importance. Equal amounts of fulvic or humic acid of soil Ml or M4 reduced the growth of

Table 4. Variance of the FOB-value in soil G TOC consumption (mg C 1-l) in culture I

II

III

FOB-value (ua kn-’ soil)

: 3 4

340 266 260 300

1250 1210 1210 1260

1820 1830 1860

41 33 32 38

Mean

291

1232

1832

s, 4.2:611.8%)

Be&ate

Bioassay for siderophores

485

Table 5. Recovery of soil supplemented ferrioxamine B. The soils were treated with ferrioxamine B (200 pg kg-’ soil). Blank soils did not receive ferrioxamine B. 1g soil was used in the bioassay Organic C-consumption (mgCl-‘) Ferrioxamine B Blank supplemented soil

Soil

Culture

Ml

I II III

0 460 1350

200 630 1600

0

207

M4

I II III

0 260 750

100 340 960

0

197

Arthrobacter similarly, ’ indicating that the origin of the soil humus compounds is not of primary importance. One could therefore expect a certain inhibition in soil suspension cultures of soils rich in organic carbon, such as soil Ml, and only a weak inhibition with soils low in organic carbon, such as soil M4. However, the evidence emerging from the experiment summarized in Table 5 indicates the opposite. The soil rich in organic C had a stimulatory effect on the soil suspension cultures, while the soil low in organic C was inhibitory. A plausible explanation for the discrepancy in the above-mentioned findings comes from the results of the experiments in z which the behaviour of ferrioxamine B in a soil suspension with soil Ml and M4 was studied (see Materials and Methods, Fig. 1 and Table 7). A large part of the ferrioxamine B is very rapidly adsorbed, particularly in the non-iron form. This is the case with both soil suspensions. However, the iron form of ferrioxamine B is less efficiently retained by the humus complex than the non-iron form. Indeed, the ratio of dissolved to adsorbed ferrioxamine B soil is for soil Ml ca. 0.1 for the non-iron form, but 2 for the iron form. For soil M4, similar trends are found. The iron form of ferrioxamine B is more extensively formed in the soil suspension with soil Ml, which is rich in organic carbon, than in soil M4 which is low in organic carbon. The dissolved iron form of ferrioxamine B is probably the most available growth factor source for Arthrobacfer JG-9. The more extensive formation of the iron form of ferrioxamine B with soil Ml in respect to soil M4 is in agreement with the higher value of EDTA extractable Fe found for soil Ml (see Table 1). The combination of a series of phenomena, namely the rapid formation of the iron form of ferrioxamine B, the fact that this form is more water-soluble in a soil suspension and finally the fact that only this form is useful for the test Table 6. Intluence of organic soil components fulvic acids, (20mg 2OOml-‘) and humic acids (1 g 2OOml-‘) and other products on the growth of Arrhrobaerer JG-9 (ferrioxamine B as arowth factor source. 2.5 UP I-‘) Treatment

Organic C-consumption (TOC, mg C 1-i)

Blank Soil Ml Fulvic acid Humic acid

638 217 70

Soil M4 Fulvic acid Humic acid Sand (1 g 200 ml-‘) N&l (1 P 2OOml-‘1

190 63 465 505

Blank

FOB value &g kg-’ soil) Ferrioxamine B supplemented soil

organism as a growth factor explains the difference in behaviour of soil Ml and M4 in soil suspension cultures. The hypothesis of sorption and, desorption of ferrioxamine B (in iron or non-iron form) is supported by additional evidence obtained in a subsequent experiment. Soils Ml and M4 (10 g) were respectively brought into contact for 30min with a 100 ml solution containing 5 mg ferrioxamine B l-l, under the same experimental conditions as described for the previous experiment. Two hundred ~1 of the filtered (0.2pm) supematant were transferred to bioassay cultures. Growth in those cultures was significantly lower than in the bioassay culture which contained 5 pg ferrioxamine B 1-l (Table 8). These facts conBnn that a substantial part of ferrioxamine B had become sorbed onto the soil-humus complex during contact with the soil. DISCUSSION

Growth of Arthrobacter JG-9 in the soil suspension culture is influenced by two major factors: siderophore concentration and iron availability. Hence the growth response of Arfhrobucrit JG-9 in soil suspension cultures containing the same amount of siderophore can be different. Iron availability in those soil suspension cultures seems to be related to the availability of the soluble ferric siderophores. The formation of the latter compounds in soil suspension cultures is most probably governed by two factors; iron extractability and sorption capacity of the

Table 7. Proportional partitioning of ferrioxamine B (380 PM) in a soil suspension (10 g soil 200 ml-‘) after 30 min contact Soil Ml ferrioxamine B Dissolved Adsorbed (W W)

Soil M4 ferrioxamine B Dissolved Adsorbed (W (W

Iron form Non-iron form

27.0 5.8

12.8 54.4

12.8 11.5

6.0 69.7

Total

32.8

67.2

24.3

75.7

Table 8. Growth in cultures of Arrhrobacter JG-9 (200 ml) which received 200 gl of a ferrioxamine B solution 5mgl-‘. The latter solution had been in contact with soil Ml(2) or soil M4(3) or was used directly(l) Treatment 1. Blank 2. Soil Ml 3. Soil M4

Ferrioxamine B conantration 5fig1-’