BIOTECHNOLOGY LETTERS Volume 17 No.4 (April 1995) pp.453-458 Received 25th January

TEMPERATURE AND DISSOLVED OXYGEN CONCENTRATION AS

PARAMETERS OF Azotobxter cbroococcum CULTIVATION FOR USE IN

BIOFERTILIZERS

Boiidar gantek * and Vladimir Marie

Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology,

University of Zagreb, Pierottijeva 6 / IV ,410oo Zagreb, Croatia.

summary

Azotobacter chroococcum was grown in continuous culture at two temperatures (30 OC and 20 OC) and different dissolved oxygen tensions (DOT) (30 % to 40 % and 70 % to 80 % of air saturation), respectively. At the temperature of 30 oC and low DOT a relatively high volumetric productivity and efficiency of nitrogen fixation were obtained. After lowering the temperature to 20 OC, an intensive formation of cysts was observed associated with a drastic decrease of the bacterial growth. Bacteria in the form of cysts kept their physiological activity for a long period of time depending on temperature and preparation.

Introduction

Azotobacter chroococcum can fix nitrogen and is therefore interesting as an ingredient of

biofertilizers. It grows from 28 OC to 32 OC (Dalton and Postgate. 1969 : Thompson. 1989a and

1989b) and growth is influenced by concentration of dissolved oxygen. High oxygen concentration

inhibits the enzyme nitrogenase and causes a decrease in the efficiency of nitrogen fixation (Dalton

and Postgate, 1969). Cells form cysts in unfavorable environmental conditions. It has been assumed

that cysts are built of alginate (Oppenheim and Marcus, 1970). The growth rate of cells is inversely

proportional to alginate synthesis. The optimal temperature for alginate synthesis is in the range of

12 OC to 18 OC (Pace and Righelato, 1980). Some components of nutrition medium such as sucrose

and metal cations can effect the formation of cysts (Foda et al., 1983). The slruclure and thickness of

the formed cysts are dependant on the iron (Fe 2+) and molybdenum (MO 6+) concentration in the

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broth (Ferrela et al., 1986). Phosphate concentration greater than 1 mm01 1-l considerably retards

the process of cyst formation (Lawson and Sutherland, 1978). The objectives of this work are : 1.

Conditions of growth and formation of cysts in cultivation of Azotobacter chroococczm for use in

fertilizers. 2. Preparation and preservation of physiological activity of bacteria for some purposes.

Materials and methods

Microorganism and media Azotobacter chroococcum (Collection of microorganisms of the Faculty of Food Technology and Biotechnology, Zagreb) was kept on Brown’s medium which contained (gl -1) : KH2PO4 0.8, MgS04. 7H20 0.2, FeSO4. 7H20 0.04, Na2MoO4 .2H20 0.005, CaC12 (anhydrous) 0.15, agar 15, glucose 5, pH 7.5 (Thompson 1989b). For inoculum propagation and continuous cultivation the modified Brown’s medium was used (gl -l) : KH2PO4 2.4, MgS04 * 7H20 0.6, FeS04 * 7H2D 0.12, Na2MoO4 .2H20 0.03, CaC12 (anhydrous) 0.45, glucose 20, pH 7.0. The media were sterilized at 121 oC for 15 min. Solutions of salts and glucose were sterilized separately and then mixed. The humus obtained after the breeding of earth-worms was used as a solid carrier for storage of the bacterial culture.

Inocul~m cukivation From slopes of Brown’s medium bacteria were seeded to 100 ml of sterile modified Brown’s medium in a 500 ml Erlenmeyer flasks and incubated on a rotary shaker (280 rpm) at 30 OC for 24 hours. 10 ml of submersed grown culture was then transferred to 90 ml of fresh medium and cultivated for another 12 hours to be used for bioreactor inoculation.

Contkhuous cultivation Bioreactor with 10 1 of modified Brown’s medium was inoculated with 5 % (v/v) of inoculum culture. The aeration was 3.5 1 m&l and agitation 250 rpm. Batch cultivation of A. ctuoococcum took 12 hours at 30 oC, when the continuous process started. The dilution rate (D) was 0.35 h-l and cultivation was performed during three residence times. After decreasing the temperature to 20 OC the dilution rate was decreased to 0.25 h-1 during the following four residence times. The dissolved oxygen tension (DOT) was 30 % to 40 % and 70 % to 80 % of air saturation depending on temperature. To keep the level of the DOT in medium at the mentioned values aeration and agitation were manually adjusted. This means that at 30 OC the aeration was 11 1 min-l and agitation 750 rpm and at 20 oC the aeration was decreased to 5 1 min-l and agitation to 400 rpm. The pH medium was maintained automatically on 7.0 by addition of 1 M NaOH or 0.05 M H2SO4.

Recovev and storage of biomass The complete bacterial culture (biomass and broth) in the form of cysts was stored : 1. at room temperature as a liquid culture and on the solid carrier (humus ; ratio : lml / 3 g ); 2. in refrigerator at + 4 oC in the liquid form. The count of viable cells (CFU) was monitored at time intervals.

Analytical procedure The DOT was measured by a galvanic oxygen electrode. The growth was monitored by dry weight measurement and by determination of bacterial cell number (CPU). The culture was centrifuged for 10 minutes at 6000 mm1 on Beckman centrifuge. Supernatants were used for glucose determination using RS-method (Dyr et al., 1963) and sediments were dried at 105 oC to constant weights. The CFU was determined by seeding the decimal dilutions of the culture on the solid Brown’s medium and incubated at 30 oC for 48 hours. The protein content in the cells was determined by Lowry

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method (Herbert et al., 1971). The carbohydrate content (%CHO) in the cells was calculated by the following equation :

%CHO=lOO- %P - %A

where is %P - percent of protein determinated by Lowry method and %A - percent of ash in bacterial cells. The formation of cysts and wall thickness were determined by electron microscopy.

Results and discussion

Effect of temperature and DOT on the growth of A. chroococcum

During batch cultivation at 30 OC a relatively long lag phase was observed (Figure 1.). Initial DOT

decreased relatively fast from 78 % to 40 % of air saturation, although bacterial growth expressed as

a dry weight and glucose consumption were hardly detectable. In the phase of intensive growth the

DOT stabilized in the range of 30 % to 40 % of air saturation. When glucose was almost depleted

from the broth the continuous flow of fresh medium started at D= 0.35 h-l. The cell concentration

remained approximately constant, the glucose concentration oscillated in the range of 2.5 - 3.0 g 1 -l,

and DOT stabilized at 30 % of air saturation.

8

7

1

0

D=O.35 h

0 2 4 6 8 10 12 Tim:‘(h)

21 24 27 30 37

Figure 1. The effect of temperature on the dissolved oxygen tension (DOT) ( m),

biomass concentration expressed as the dry weight ( D.W. ) ( A), ccl1 number

(CFtJ /ml) (0) and glucose concentration (S) (A) during cultivation of A. chroococcum

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After three residence times the temperature was lowered to 20 oC and the dilution rate was decreased

to 0.25 h -l to prevent the washout of the culture caused by the shift of growing conditions to

suboptimal values. The new steady state was established as well as the new DOT value (70 % to

80%). The decrease of bacterial growth was evident as the curves for the culture optical density and

glucose consumption show. The results were expected because decreased temperature caused the

increase of DOT. Consequently, the effect of the temperature on the growth was difficult to separate

from the effect of increased DOT.

EYEciency p of cultivation

In continuous cultivation performed at 30 oC (D= 0.35 h-1) a relatively high volumetric productivity

(1.12 g 1 -lh -1) was obtained (Table 1.). The efficiency of nitrogen fixation (YN,s) was 0.0192 g N/ g

glucose which corresponds to the literature data (Tchan and New, 1986).

Table 1. Efficiency parameters in continuous cultivation of A. chroococcm.

T (OC) D (h -1) (g 12h-l)

YNIS y x/s (g Nz/g glc ) (g g -9

30 0.35 1.12 0.0192 0.20

20 0.25 0.42 0.0118 0.28

where T is temperature, D is dilution rate, Pr is volumetric productivity, YN,~ is efficiency of

nitrogen furation and Ym is biomass yield based on glucose.

However, the relatively low biomass yield based on glucose (Yx,s = 0.2 gg -l) could be a

consequence of tbe large energy consumption needed for glucose degradation, bacterial growth or

nitrogen fixation. Lowering the temperature to 20 OC caused an increase of DOT and biomass yield

based on glucose (0.28 g g -‘), but also decrease of the volumetric productivity (0.42 g 1 -lh -l) and

efficiency of the nitrogen fixation (0.0118 g N/g glucose). This was a consequence of unfavorable

environmental conditions and the formation of cysts. Therefore, the correlation between the bacterial

dry mass and the number of the cells became more irregular (CFU curve).

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Formation of cysts

The curves in Figure 1. show the relationship between the cell number (CFU) and dry weight of

biomass in 1 ml of culture. In the steady state of continuous culture at 30 oC the bacterial number

was around 108 CFU per ml and biomass dry weight was 3 to 3.5 mg ml -1. Accordingly, the

calculated average mass of single bacterial cell was about 3.25 . 10-8 mg. Lowering the temperature

to 20 oC caused the decrease of the bacterial number to 107 CFU per ml and biomass dry weight to

1.6 to 1.7 mg ml -l and increase the average mass of single bacterial cell to 1.65 . 10-7 mg.

Accordingly, oscillation of CFU was a consequence of aggregated cysts and their liability to

mechanical damage (Tchan and New, 1986) and dots not indicate the changes caused by the lower

temperature or increased DOT. As seen in Table 2. the biomass grown at 20 OC had a high

carbohydrate content (75.5 %) and biomass grown at 30 oC had a relatively high protein content

(45.9 %). During intensive growth at 30 oC cells had thin cell wall (20 nm), while at 20 oC cell wall

was much thicker (60 run). Thcrcforc, the protein content of cells dccrcased from 45.9 to 22.5 % and

carbohydrate content increased from 52.1 to 75.5 %.

Table 2. Content of bacterial biomass and cell wall thickness of A. chroocuccurr~ at different

dilution rates during continuous cultivation.

D(h-‘) % Protein 8 Carbohydrate % Ash Cell wall thickness

(nm)

0.25 22.5

0.35 45.9

where D is dilution rate.

75.5 2 60

52.1 2 20

Preservation of physiological activity of A. chroococcum

The liquid culture stored at room temperature and in refrigerator kept its physiological activity 21

and 50 days, respectively. When the culture was stored on stcrilc carrier at room temperature the cells

kept its viability over 70 days (Figure 2.).

On the basis of these results we recommend the following procedure for the production and

preservation of A. chroococcum as a component of biofcrtilizers :

1. Cultivation at optimum temperature and DOT for the growth (30 oC and 30 % to 40 8 of air

saturation).

2. Decrease of the temperature to 20 OC several hours before the harvesting of the cells.

3. Storage of the cells on the humus at room temperature.

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1

0 7 14 21 26 36 44 Stora~ time &ys)

66 70

Figure 2. Change of cell numbers in biomass from continuous cultivation stored as a liquid

culture at + 4 oC ( 0 ) and stored at room temperature as a liquid culture ( A )

and on the solid carrier ( l ).

References

Dalton, H. & Postgate, J. R. (1969), Journal of General Microbiology, 54,463 - 473. Dyr, J. Gregr, V. & Seiler, A. (1963), Lichvarstvi, Statni nakladatelstvi technicke Literatury Praha pp. 147 - 157. Ferrela, N. F. Champlin, A. K. & Fekete, F. A. (1986) FEMS Microbiolology Letters 33, 137 - 142. Foda, M. S. Naguib, A. I. Shawky, B. T. & Rizhallah, L. A. (1983), Zentralblatt Mikrobiologie 138, 135 - 144. Herbert, D. Phipps, P. J. & Strange, R. E. (1971), Chemical analysis of microbial cells. In : Methods in Microbiology, Norris, J. R. & Ribbons, D. W. eds. vol 5B. pp. 243 - 265, New York : Academic Press. Lawson, C. J. & Sutherland, I. W. (1978), Primary Product of Metabolisam, Rose, A. H. eds. pp. 328 - 389, New York : Academic Press Inc.. Gppenheim, J. & Marcus, L. (1970), Journal of Bacteriology 101,286 - 291. Pace, G. W. & Righelato, R. C. (1980), Advances in Biochemical Engineering 15,41 - 71. Tchan, Y. T. & New, P. B. (1986), Azotobacteraceae. In : Bergys Manual of Systematic Bacteriology, Krieg, N. R. & Holt J. G. eds. pp. 219 - 230, Baltimore : Williams & Wilkins. Thompson, J. P. (1989 a), Plant & Soils 117,9 - 16. Thompson, J. P. (1989 b), Plant & Soils 117, 17 - 29.