Microbiological examination of some Sudanese soils

Zbl. Bakt. II. Abt. 134 (1979), 536-543

[From the Department of Microbiolcgy, Faculty of Agriculture, Cairo University, Giza, Egypt]

Microbiological Examination of some Sudanese Soils N. A. HEGAZI and SANEYA AYOUB With 6 Figures

Summary The composition of the microflora in different layers of four representative soil profiles of Sudan were studied. Counts of all types of microorganisms decreased significantly with depth in soil. Azotobacter, in particular, occurred in high densities; representative strains were isolated and studied for their different characteristics. Beijerinckia was detected as well, and a new method for the estimation of their numbers in pure cultures, based on the overlay agar technique, is described.

Zusammenfassung Die mikrobiologische Analyse einiger sudanesischer Boden zeigte eine hohe Azotobacter-Dichte. Beijerinckia wurde in einer von 12 Proben nachgewiesen. Eine neue Methode, Beijerinckia-Zellen in Reinkultur zu zahlon, die auf der Zweischicht-Agar-Technik (overlay agar) beruht, wird beschrieben.

The last two decades have witnessed investigations, recently reviewed by ABD-ELMALEK (1971), who showed that the Nile Valley soils harbous exceptionally high Azotobacter populations. Examined soils from neighbouring countries (HEGAZI 1969; ISH'AC et al. 1970) indicated that the abundance of such organisms is a common phenomenon in North African and Middle Eastern countries. The present work introduces further information on general microbial analysis and occurrence of asymbiotic N 2-fixing bacteria in soils of one more country of this area, i.e., Sudan.

Materials and Methods Samples 12 samples, representing 4 soil profiles, were obtained from various localities (Table 1). All soils were passed through a 2 mm sieve. 1: 9 (w/v) soil in water suspension was prepared; these suspensions were used for 10-fold dilution series in tap water.

Counting methods 1. Dilution plate count. The dilution plate count is widely used for estimating the total bacterial count in soil (PARKINSON 1971). 1 % soil extract agar medium amended with 1.0 g glucose and 0.5 gK 2 H P 0 4 per litre was used. Colonies were counted after 14 days of incubation at 30- 32°C. 2. Dilution-tube methcd was used for the estimation of numbers of ammonifiers, denitrifiers, proteolytic and anaerobic spore-forming bacteria. Media and techniques applied were those of

Microbiological Examination of some Sudanese Soils

537

Table 1. Some characteristics of the soil samples examined Depth (em)

pH

Soil texture

Moisture content

I. Zeidab

0- 25 25- 45 >45- 85

8.8 7.9 7.5

Sandy clay

3.4 :3.9 4.7

II. Habila

0- 20 55-110 > 110-160

7.4 8.1 8.0

Clay

6.0 7.0 7.4

0- 30 50- 75 > 75-115

9.0 7.5 7.1

Sandy clay

4.6 5.9 7.4

0- 20 >20- 50 >50- 95

7.8 7.7 8.1

Clay

8.5 8.9 9.0

Locality

III. Goz-el-Rehid

IV. Southern Fung

PoCHON and TARDIEUX (1962) and PoCHON et al. (1969). MPN estimates were derived from MCCRADY'S Tables (1918) after 15 days of incubation at 30- 32°C.

3. Azotobacter counts were obtained by both techniques of surface-inoculated plates and membrane filters (HEGAZI and LEITGEB 1973; HEGAZI and NIEMELA 1976). 4. Beijerinckia was detected in liquid enrichment cultures, prepared according to BECKING (1961). The overlay agar method used in phage research was applied for plating Beijerinckia. Well aerated cultures of Beijerinckia (continuously shaken or bubbled with air) were first shaken vigorously for 5 minutes. Then aliquots (0.5 ml) of suitable dilutions were mixed with a 2-ml semi-solid medium (containing 0.6 % agar) after being molten and brought down to 45°C. The mixture was immediately poured and spread on the surface of solidified agar base-layer in Petri dish. Well developed and limited colonies were obtained on incubation for 4-7 days at 30-32 "C (Fig. 6).

Chemical methods 1. Total nitrogen content of bacterial cultures was determined by the conventional macroKjeldahl method (BREMNER 1965).

2. Total organic carbon in bacterial cultures was determined by the Black and Walkley rapid titration method (PIPER 1950). 3. Glucose was determined colorimetrically by the method of SOMOGYI (1952).

Electron microscopy Mounts of Azotobacter and Beijerinckia cells were negatively stained with phosphotungstic acid (2 %, wjv, pH 6.8). Thin sections were prepared from Beijerinckia cells and mounted on formvar-coated grids (HEGAZI and CIAMPOR 1976). The ultrathin sections were doubly stained with 2 % (wjv) uranyl acetate and lead citrate (VENABLE and COGGESHALL 1965). Electron micrographs were taken with a JEM 6c electron microscope at 80 kv.

Results The results of quantitative microbial estimations are given in Fig. 1. The concentration of microorganisms was greatest in the upper soil layers and decreased markedly with increasing depth. Total bacterial numbers exceeded those reported for the different bacterial groups, except for ammonifiers in few cases and particularly in surface soils. This might be attributed to differences between methods applied, i.e., the plate count and the dilution-tube method. In addition, the partial anaerobiosis developed

538

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Fig. 1. Counts of various bacterial groups obtained for different soil samples.

Table 2. Counts of Azotobacter, obtained by the method of surface-inoculated plates (SP) and membrane filter technique (MF) Locality

Number of Azotobacter (x I02)/g oven-dry soil Surface soil

I II III IV ND

=

Sub-soil

Deep soil

SP

MF

SP

MF

2725 315 286 208

ND ND ND ND

418

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not determined, -

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14 3 4

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MF

15 2 I I

Microbiological Examination of some Sudanese Soils

539

Fig. 2. Electron micrograph of 24-h-old cells of A. chroococcum, negatively stained with phospho tungstio acid ( X 30,000 magnification).

Fig. 3. Typical appearance of 14-day-old B. indica plate culture.

540

N.

A. HEGAZI

and

SANEYA AYOUB

4a

Fig. 4. Electron micrograph of 7-day-old cells of B. indica, negatively stained with phosphotungstic acid (a, X 38,500; b, X 26,250 magnification).

in the liquid cultures, following the latter method, encourages the growth of facultative anaerobic bacteria better than the strict aerobic conditions found on the surface of agar plates, used for total bacterial counts. Although the method applied for the detection of nitrifying bacteria is not sensitive enough, it is generally concluded that the Nitrosomonas group is more confined to the soil surface than the Nitrobacter. Azotobacter was prevailing in surface soil, and the low densities found in deep soils were only detected by the membrane filter technique (Table 2). 19 representative strains were isolated from various localities at different depths. They belonged to A. chroococcum and their characteristics were found to be in good agreement with the descriptions of Beijerinck (1901), JENSEN, H. L. (1954), and JENSEN, V., and PETERSEN (1954). Their typical cell morphology is presented in Fig. 2. The predominance of the particular species A. chroococcum was previously reported in soils of many other countries (MISHUSTIN and SHILNIKOVA 1971). All strains were able to utilize a wide range of carbon sources. Glucose, ethanol, and sucrose mannitol were utilized best; growth on starch was very much delayed. Rhamnose gave no or trace growth (JENSEN, V. 1961). The strains fixed significant quantities of nitrogen (10 -16 mg N assimilated/g consumed glucose) when grown in N-deficient liquid medium for 14 days at 30 -32 "C. Beijerinckia was detected in the only surface soil of the locality Habila. A typical strain (Fig. 3), belonging to the species B. indica (BECKING 1974a and 1974 b), was

+

Microbiological Examina tion of some Sudanese Soils

541

4b

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Fig. 5. Section of a cell of B . i ndica ( X 30,00 0). Note the large li poid g ran u le (L ) a t b oth poles of the cell.

542

N.

A. HEGAZI

and

SANEYA AYOUB

obtained. The characteristic large lipoid bodies at each end of the cell are very well distinguished in negatively stained preparations (Fig. 4) and in ultrathin sections (Fig. 5). The ability of this strain to fix molecular nitrogen was estimated to be in the range of9-11 mg N/g consumed glucose when grown in N-deficient acid medium for 16 days at 30-32 "C.

Discussion The environment has a major influence on the microbiological composition of a given ecosystem. The relatively high temperature and the type of vegetation ensure higher microbial activity for larger periods of the year in the soil of tropics, subtropics, and semi-arid regions than in temperate regions. A unique property of the soils of the former regions is the acquisition of high populations of free-living N 2-fixing bacteria of the genera Azotobacter and Beijerinckia (HEGAZI 1969, ABD-EL-MALEK 1971, BECKING 1961). The results presented here show that the Sudanese soils are no exception. These soils belong to the category of active soils being able to yield 2000 Azotobacter coloniesjg soil (WINOGRADSKY 1926). Azotobacter was found to occur at all depths examined down to 160 em, provided the membrane filter technique was applied. Such technique has been recommended for counting Azotobacter in various habitats, particularly those having low densities of the organism (HEGAZI and NIEMELA 1976). The soils of Sudan were not included in the comprehensive survey of BECKING (1961) on the occurrence of Beijerinckia in the soils of different parts of the world. The accidental detection of such organism, although present in one out of 12 samples, show the necessity of searching for a rather adequate and sensitive enrichment technique and medium for the detection of these bacteria in soil. In addition, the lack of

Fig. 6. 7-day-old colonies of B. indica, grown with the overlay agar technique, using thick (left) and thin (right) basal layer of agar.

Micr ob iological Examination of some Sudanese Soils

543

a convenient metho d for count ing Beij erinckia cells does not encourage studies on growth kinetics of the organism. The application of the overlay agar techniqu e used in phage assay. as described in Materials and "Methods, proved satisfact ory . Well developed and limit ed colonies were successfully obtained after 4 -7 days of incubation at 30-32 00 (Fig. 6). A c know l e dge me n t " re t hank Dr. F . CIA:>IPOR of t he I nstit ute of Virology , Slovak Academy of Scien ces , Brati slava, for hi s coo pe ration with e lec t r on mi cr oscop y.

References ABD·EL. MALEK, Y . : Free -li ving n itrogen- f ixing ba cteri a in E gyp t ia n soils a n d t he ir po ssi bl e cont ribution t o soil fer tilit y . Pl ant an d So il, Specia l vo lume (1971), 423-442. BEeKING, J . R.: St ud ies on nitrog en-Ii x ing bacteria of t he gen us B eij erin cki a. 1. Geograph ical a nd eco log ica l distributi on in soi ls. P la n t and Soil 14 (1961 ), 49 - 8J. F amily Az otobaetcriaceae. I n : Bergey's Determinative Bact eri ology . 8th ed. (BUCHANAN, R. E ., a nd GIBBONS, N., eds.) The Will iams and Wilkins Company, Balt im or e I974a, 253-26J. Nitrogen-fixing bacteria of th e gen us B eijerinckia. Soil Sci. 1I8 (1974b), 196-212. BEIJERINCK, M. W.: Uber oligo n itrophi le Mikroben. Zbl. Bakt. 117 (1901) , 561-582. B REMNER, J. M.: Organic forms of ni trogen . In : Methods of Soil Anal ysi s. Agr onomy 1965, 1238 t o 1255. HE GAZI, N . A. : Aerobi c n on- symbi ot ic N 1 ·f ix in g bact eria in E gyp t . l\I. Sc. Thesis, Faculty of Agricul t ur e, Cai ro U n iversity (1969 ). Hl:GAZI, N . A ., a n d LEIT GEB, S. : The a p plicatio n of the m embrane fi lt er t echnique in co unting A zotobacter. Zbl. Bakt, II 128 (1973) , 624- 628. and CIAMPOR, F .: An electron mi cro scope s t u dy of t he lyti c cycle of A zotobacter bacteriophages . Soil Bi oI. Biochem. 8 (1 97 6), 365 -368 . a n d NIE~IEL.~ , S. : A no te o n t he estima t ion of A zotoba-eter dens ities by m embrane fil ter tee hniq ue . J. Appl. Bact. 41 (1976), 3 11 -3 13. I SHAC, Y . Z. , KA DDOURI, N . S., and Y OUSEF, A. N.: Den si t y of Azotobacter in so me southe rn part s of Iraq. Soc. Ap p l. Bact. ; 2nd Con f., Ca i ro 1970. J ENSEN, H . L.: The Azoto ba cter-iacea e, Ba ct. R eviews 18 (1954 ), 195 -2 14. JENS EN, V.: Rhamnose for d et e ction a nd isol a tion of A zotobacter v i11eland ii Lipman. Natu re (Lo n don) 19 ( 1961), 83 t -833. - a n d PET ERSEN', E. J. : Stu di es on t he occ urrence of A zotobacter in Danish forest soils. Royal V et . a nd Ag r ic. College, Copen hagen, Ye arbook 1954, 95- 108. MCCRADY, M . R. : Tables for r a p id in t erpretati on of ferment a t ion t ube res ults. Ca n . Publi c H ea lth J . 9 (1918 ), 275. MISHUSTIN, E. N., and SHIL'NIKOVA , V . K . : Bi ological Fixation of Atmospheri c Nitrogen. Ma cmillan 1971. PARKINSON, P., GRAY, T. R . G., and WI LLIAMS, S. J.: Metho d s for Studying the Ecology of Soil Micr oorganisms. IBP Handboo k No. 19, Blackwell Scientifi c P ub licat ion s, Oxford and Edinbu rg h 1971,57-70. PIPE R, C. S. : Soil and Pl ant Anal ysi s. I n t erscience Publish er s Inc. , New York 1950. P OCHON, J. , a n d TARDIEUX, P. : Te ch ni q ues d 'analyse en m icr ob iologi e du so l. Edit ions d e la T oure lle . St. Mande, F r anc!', 196:!. - - and U'A GUILAR, J . : Method ological pro blems ill so il biology. In: So il B iolog y , UN ESCO R e vi ew of R esearch ; Natural R eso urces R esearch No. IX (1969),1 3- 63. S01\IOGYI, I\L: No te s on sug ar determi nation. J . Biol , Che rn. 195 ( 1952), 19-23. VENABLE, J. R ., a nd COGGESHALL, R. : A simpli fied lead citrate s t a in for use in electro n m ic roscop y. J. Cell BioI. 25 ( 1965), 407 - 408 . \VIXOGRADSKY, S .: E tude S U I' la mi crobiologic du so l. II. Sur les microb es f ix ateurs d 'azot e. An n . I n st. P ast eu r (Fran ce ) 40 (1926), 455 - 520. Au thors ' address : Dr . N . A . H E;GAZI and SA!>EYA AYO UB, D ep t . of Microbiol ogy , Faculty of Agric u ltu re, Cai ro Univers ity , Giza, E gypt. 35 a