ON THE USE OF FROTH FLOTATION IN THE RECOVERY OF Bacillus sphaericus SPORES

ON THE USE OF FROTH FLOTATION IN THE RECOVERY OF Bacillus sphaericus SPORES

E.M. RIOS ^{1, *} , C.E. LOPES ¹ and F.P. de FRANÇA ²

¹ UFPE - Dept. de Antibióticos, Centro de Ciências Biológicas, Av. Prof. Morais Rego, s/n,

CEP 50.670-901, Recife PE, Brasil. Phone/Fax: [phone]

² UFRJ - Escola de Química, Centro de Tecnologia, Bloco E, sala E-104, CEP 21.941-900,

Rio de Janeiro RJ, Brasil. Phone/Fax: [phone]

(Received: September 3, 1996; Accepted: March 4, 1997)

ABSTRACT - The recovery of Bacillus sphaericus strain 2362 spores from fermented medium by batch flotation was tested under different conditions. Flotation kinetic studies were performed at 800 rpm and 3 l air/min. The pH values were adjusted at the following set of values: 5.0, 7.0 and 9.0. The results showed that the spore removal rate is influenced by the pH value. At pH equal to 5.0 we observe an adverse effect on the spore concentrate obtention. In this situation the maximum value of the concentration factor was 1.4 when the recuperation percentage was 99%. At pH equal to 7.0 the concentration factor reached the highest value, 7.0, but the recuperation percentage stayed around 96%. Field experiments with the floated material demonstrated that its larvicide activity was sufficient to keep a Culex quinquefasciatus larvae population under control at in a breeding site, during 3 months with 2 applications.

KEYWORDS: Bacillus sphaericus, bioinsecticide, cell recovery, flotation.

INTRODUCTION

The worldwide consumption of insecticides has revealed the clear trend to substitute nonselective chemical products by products of biological origin specifically directed toward the target species. Of these biological agents, the one based on entomopathogenic bacteria represents 92 % of the bioinsecticides sold for agricultural and public health use (Keller and Becker., 1994). This procedure for insect control used protects both the ecological environment and human health.

Bacillus sphaericus is an entomopathogenic bacterium considered to be a highly selective agent for mosquito control mainly active against Culex larvae but also against larvae of other disease transmitting insects.. An advantage of the Bacillus sphaericus, as compared to B. thuringiensis var israelensis, is its higher capacity to recycle itself in high organic content aquatic systems such as sewers, water treatment effluents, irrigated pastures and rice fields (Silapanuntakul et al. 1983; Mulla et al. 1990).

Flotation is a chemical engineering unit operation commonly used in mineral ore separation / concentration which is being increasingly used in biotechnological processes (Lopes et al. 1993; Nunokawa et al. 1971; Santana, 1994). The tendency of the Bacillus sphaericus spores to adhere to the surface of the air bubbles and to concentrate in the foam is clearly observed (Rios et al. 1992). The separation of different Bacillus species spores (anthracis, cereus and subtilis) through the foaming of the fermenting medium is also reported by other authors. (Boyles and Lincoln, 1958; Gaudin et al. 1960).

The aim of this paper is to study the use of the separation and concentration of the Baccilus sphaericus spores by froth flotation in an Outokumpu flotation cell.

MATERIAL AND METHODS

Microorganism

The Bacillus sphaericus strain 2362 was used. The microorganism was maintained as spores on the surface of filter paper discs which, after the application of 50 m l of a microbial suspension of known concentration (1.44 x 10⁹ spores. ml^-1 ), were dried at ambient temperature (28 ± 1^oC) and kept under refrigeration (5 ± 1^oC).

Nutrient Media

Nutrient agar was used in spore quantification in the samples of fermented medium and in those of solid wastes. This was also the nutrient medium for production of spores to be applied on paper discs for culture conservation. The biomass production medium contained, in g.l^-1: caseine peptone 10.0; yeast extract 1.0; KH₂PO₄ 1.0; MgSO₄.7H₂O 0.1; CaCl₂.2H₂O 0.1; FeSO₄.7H₂O 0.01; MnSO₄.H₂O 0.01; ZnSO₄.7H₂O 0.01. The pH was adjusted to 7.2 using NaOH 40% solution. This medium was also used for pregermination.

Inoculum

The inoculum was prepared using Fernbach flasks containing 250 ml of medium. The preparation was initiated from a paper disc impregnated with a known concentration of spores per flask. The flask was shaken at 150 rpm and 28 ± 1^oC during 12 to 15 hours. The inoculum size was 5 % of the final volume of the fermentation.

Biomass Production

The microorganism production was made in batch, using agitated Fernbach flasks with 250 ml of medium at 28 ± 1^o C during 48 h.

Hydrodynamic profile

A few tests were carried out with the fermented medium to define the system’s approximate hydrodynamic profile. In these tests, the agitation rate was varied from 0 to 1600 rpm and the aeration flowrate from 0 to 10 l air/min. Visual observations were made regarding the medium hydrodynamic behaviour under agitation and aeration.

Flotation

All flotation experiments were carried out in triplicate, using an Outokumpu flotation cell with 1.5 l capacity. Foaming agent was not added, nor was the liquid volume complemented during the experiment. A flotation time of 15 min. was used for the agitation and aeration experiments; in those where the nutrient media were adjusted to different pH values: 5.0, 7.0 and 9.0, a flotation time of 7.5 min. was adopted.

The flotation experiments were carried out at 640, 800, 960, 1120 and 1280 rpm, each of these under three different aeration conditions, 3, 5 and 7 l air.min^-1. In the experiments with different pH values, the nutrient media were adjusted with 1:2 diluted H₂SO₄. The volumes added were recorded, with the same volume taken out of the adjusted medium volume to be floated so that the sample volume was kept at 1.5 l.

Spore Quantification

Spore concentrations were evaluated by colony count after a sample thermal shock at 80^oC for 12 min., and expressed as CFU.ml^-1. The colonies formed on dishes with nutrient agar, incubated at 30 ^oC, were counted after 20 to 22 hours. For statistical evaluation, each sample dilution had six replicates on different Petri dishes. The experimental results were expressed as spore concentration factor

spores per unit volume in flotattion froth

spores per unit volume in the original culture

and as spore recovery percentage

spores in flotation froth

spores in the original culture

x 100

The first parameter gives an idea of increase spore concentration in the flotation froth as compared to the original culture. The second expresses the spore removal efficiency.

Biological Test

The larvicide activity of the floated material was evaluated on populations of Culex quinquefasciatus larvae in the 3rd and 4th instars. The selected breeding site for the biological test was a covered area measuring 5.4 m². The treatment consisted of two applications of the material (containing approximately 1.39 x 10⁹ CFU/ml) over a 3 month period, in a 1 ml.m^-2 proportion.

RESULTS AND DISCUSSION

Figure 1 shows the approximate hydrodynamic profile of the system. Five phases were observed: Phase I, laminar regime with sparse bubbles; Phase II, laminar regime with full bubbling (well distributed bubbles); Phase III, turbulent regime and sparse bubbling; Phase IV, turbulent regime with full bubbling and Phase V, with unstable functioning, presenting liquid washout in the foam and spilling. The flotation experiments were carried out under the hydrodynamic conditions represented by Phase IV, considered adequate for flotation purposes due to uniform bubbling under turbulent conditions. The selection of this region excludes from the experiments the minimum extreme agitation and aeration conditions (640 rpm at 3 l air/min ) and the maximum extreme conditions (1280 rpm at 7 l air/min).

Figure 2 shows the results otained for spore concentration factors as a function of the agitation and air flowrate. At agitation rates of 800, 960 and 1120 rpm a clear trend toward the reduction of the concentration factor as the air flowrate increase is noted. In this figure it is possible to perceive that the best recuperation percentages occurred at 3.0 l air/min. At the extreme conditions no influence of the aeration flowrate on the concentration factor was perceived. A significant reduction of it was observed at the agitation rates of 1120 and 1280 rpm for all values of air flowrate.

An analysis of Figure 3 showed that the liquid pH values had influenced the flotation process and the spore concentration was proportional to the pH increase. This work also shows a strong effect in the spore separation at pH = 9. The maximum spore concentration occurred around 7.5 minutes. The curve profile reveals that the flotation froth is becoming diluted after this time.

Figure 4 presents strong variations in the flotation froth concentration profile at different pH values. This results does not fully corroborate the results obtained by Boyles and Lincoln (1958) which found no significant pH influence on the spore collection at pH values 6.0, 7.0 and 8.0.

Figure 5 points out the occurrence of a large liquid washout at pH = 5.0. At this pH value the bubbles were small and presented a wetted and bright aspect which reveals an excess of liquid washout with low spore concentration. The profile of the curves in Figure 6 confirm this observation. In spite of recuperation percentages above 90% the concentration factors were substantially different.

The field experiments carried out with floated material showed a 100 % reduction in the population density of the 3rd and 4th instar Culex quinquefasciatus larvae. (See Figure 7). The second application of the biologically active material was made 36 days after the first, when the larvae density reached 37.2 % of the original value. Control of the Culex larvae population at the breeding site was still maintained 56 days after this second application.

Microscopic observations of the floated material showed that they did not present cell or spore agglomerations, as observed in the fermented material. Macroscopically the material can be seen as a light suspension, with little tendency to form a compact sediment.

Figure 1: Hydrodynamic profile of the batch flotation process of B. sphaericus strain 2362 in peptonated medium without foaming agent. Outokumpu flotation cell with 1.5 l Phase I, laminar regime; Phases II and III, turbulent regime; Phase IV, turbulent regime with full bubbling; Phase V, unstable functioning.

Figure 2: Effect of agitation and aeration on spore concentration factor.

Figure 3: Spore concentration in flotate, as a function of flotation time under different pH values.

Figure 4: Spore concentration factor as a function of flotation time at different pH values.

Figure 5: Flotation froth volumes as a function of flotation time at different pH values.

Figure 6: Relationships between spore recovery percentage and spore concentration factor in flotation.

Figure 7: Populational larvae density profile of Culex quinquefasciatus treated with floated material based on B. sphaericus.

CONCLUSIONS

The results of this work allow one to conclude that agitation rate values of 800 and 960 rpm at 3 l air/min are adequate for Bacillus sphaericus spore recovery from peptone-based culture media. No foaming agent has to be added.

The flotation process allowed recuperation percentages above 94% and a concentration factor equal to 5.8 at pH = 7.0 and flotation time of 10 minutes. Bigger recuperation percentages (98.2% and 99.1%) were obtained at this flotation time but the concentration factors were smaller (4.1 and 1.37) for values of pH equal to 9.0 and 5.0, respectively.

Flotation was revealed to be a promising operation for separating and concentrating the Bacillus sphaericus spores, since it is a simple, low-cost process, viable for obtaining spore concentration adequate for the preparation of bioinseticide formulations.

The flotation froth physical characteristic of not showing flocs or compact sediment can be considered to be an advantage for formulated materials preparation.

ACKNOWLEDGEMENTS

The authors express their thanks to the Departamento de Entomologia of the Centro de Pesquisas Aggeu Magalhães, in particular to Dr. Leda Regis for carrying out the field experiment with our material; to the Laboratório de Tecnologia Mineral of the Universidade Federal de Pernambuco which allowed the use of the flotation unit; and to the Fundação de Amparo à Ciencia e Tecnologia do Estado de Pernambuco and the Conselho Nacional de Pesquisa for the financial support.

NOMENCLATURE

CFU Colony Forming Unit

% Rec Spore recovery percentage = (Vo-Vw).Xs.(Vo.Xso)^-1.100

Vo Volume of sample to undergo flotation

Vw Volume of waste (ml)

Xs Spore concentration in flotation froth (CFU/ ml) = ((Vo.Xso) - (Vw.Xsw)). .(Vo-Vw)^-1

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*

To whom the correspondence should be addressed

Publication Dates

• Publication in this collection
  09 Oct 1998
• Date of issue
  June 1997

History

• Accepted
  04 Mar 1997
• Received
  03 Sept 1996