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Revista de Microbiologia

Print version ISSN 0001-3714

Rev. Microbiol. vol. 29 n. 4 São Paulo Oct./Dec. 1998 



Rosana Ferreyra; Graciela Lorda and Antonio Balatti* 
Departamento de Química, Facultad de Ciencias Exactas y Naturales. UNLPam. Santa Rosa, La Pampa, Argentina.

Submitted: November 27, 1997; Returned to authors for corrections: April 24, 1998;
Approved: September 17, 1998




The influence of aeration and automatic pH control on the production of a-amylase by a strain of Bacillus subtilis NRRL 3411 from acid cheese whey was studied. Tests were carried out in a rotary shaker and in mechanically stirred fermenters. a-amylase was analysed according to DUN’s method. Oxygen absorption rate was determined by Cooper’s method. Cell oxygen demand was determined as oxygen consumption in a Warburg respirometer. The level of dissolved oxygen was measured by means of a galvanic silver-lead electrode. Results suggest the possibility of industrial use of acid cheese whey as a carbon source for a-amylase production, since the yield was similar to that produced with lactose. The highest a-amylase levels 10,000 DUN/ml units were not attained at higher aeration rates -431 mLO2/L.h-. The indicated value correspond to a 96 h process with automatic pH control at 7.5. These conditions resulted in double concentration of a-amylase. The enzyme production was directly related to growth in the form of cell aggregates.

Key words: Bacillus subtilis, a-amylase, acid cheese whey, automatic pH control.




The microbial amylases have found wide scale industrial application. These in enzymes are used in starch-processing, baking and in the textile industry.

The production of a-amylase, using strains of Bacillus, is influenced by both nature and concentration of carbon and nitrogen sources. It has been shown that Bacillus subtilis NRRL 3411 produces higher enzyme concentration in a lactose medium than in starch, saccharose or glucose. (6)

Since lactose is the most expensive of these carbon sources, we considered the possibility of using acid cheese whey, a by-product of the dairy industry.

Acid cheese acid whey is a readily available substrate and a powerful poluent. Its use in the production of liquid stabilized enzyme preparations could represent a way of reducing pollution whilst providing a low cost carbon source medium at industrial level.

The aim of this work is to improve the production of a-amylase. Using a previously selected culture medium, a comparative study was carried out in Erlenmeyer flasks and in a fermenter at different aeration rates. Enzyme production at different pH values was studied and an automatic pH control process developed.



Microorganism. A strain of Bacillus subtilis NRRL 3411, maintained as spores in peat (see Table 1), was used. Stock cultures were prepared as follows: one part of river sand previously washed, pH adjusted to 6.8-7, was thoroughly mixed with high water holding capacity peat (100%) 100 g of this peat can absorb 100 g of water, and yet remain as a fine dust with all its characteristics. The mixture was adjusted to 12% humidity content, and sterilized in five-gram portions, for 3 hours at 121ºC. The mixture was then impregnated with a 3 ml spore suspension in medium 2 (see Table 1). Tubes were hermetically sealed with either a threaded cap or a plastic film cover over cotton caps, and stored at 5ºC.


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Media. Culture media are shown in Table 1. Experiments were carried out using lactose or acid cheese whey as a carbon source (6).

Inocula. Each flask was seeded with a peat-kept spore suspension in 5 ml sterilized distilled water, previously exposed to a 10 min 100ºC thermal shock. (3,6,12)

Cell growth. Microbial growth was quantified by optical density measurements (650 nm) and by dry weight. A 10 ml sample was centrifuged at 2,200 g for 20 min. Precipitate was washed with distilled water, resuspended in water and dried at 100ºC until constant weight was reached.

Determination of a-amylase. DUN’s method was used to determine the a-amylase activity of cultures: 10 ml of soluble starch solution at 1% were added to 1 ml of the cell-free solution (7.2 pH), and incubated at 40ºC for 10 min. The enzyme reaction was stopped by adding 10 ml HCL 0.1N solution. 1 ml of this solution was then added to 10 ml of iodized iodine solution. The optical density was measured at 660 nm. A blank, substituting the enzyme solution for distilled water, was carried out. One DUN enzyme unit is defined as the quantity of enzyme that causes 1% reduction in the intensity of the blue colour obtained by mixing the iodine solution and the starch solution at 40ºC for 1 min. (1).

Consumption of carbon source. To determine lactose concentration in the culture medium, Miller’s spectrophotometric method (8), which measures reducing sugars, was used. A standard curve was obtained with lactose concentration between 50 and 330 ppm, using a 550 nm wavelength.

Cell oxygen demand. Cell oxygen demand was determined by means of a Warburg respirometer at 28ºC. The variation in the gas quantity is measured at constant volume. Any change in the gas quantity is measured by a pressure change in a manometer (11).

Oxygen absorption rate. The oxygen absorption rate (OAR) was measured using the sulphite method (2). The oxygen absorption by solution of sodium sulphite with cupric ion as catalyst may be used as measures of oxygen solution rate. The principle of such method is that the rate of oxidation of the sulphite is limited only by the rate of the oxygen transfer from gas to liquid.

Dissolved oxygen. Dissolved oxygen was measured with a sterilizable silver-lead galvanic electrode.

Operating conditions. Inocula were produced in 250 ml Erlenmeyer flasks containing 50 ml of medium Nº 2. Production was carried out in 500 ml Erlenmeyer flasks containing 100 ml of medium Nº 3 in a rotary shaker at 250 rpm and 2.5 cm eccentricity. For the preparation of the culture media, the medium components peptone, yeast extract, ammonium sulphate, phosphates and sulphates were sterilized separately of the carbon source and carbonates, at 121ºC for 20 min. After sterilization all the components were mixed. It was necessary to sterilize the medium components separately to avoid any alteration and/or interaction which might modify the pH in a significant way.

Experiments were carried out in media containing either lactose or whey, with concentration expressed as lactose at 30 g/l. The whey used was a by-product from mozzarella cheese production. This raw material was provided by Cooperativa Láctea (a dairy products manufacturing plant) Santa Rosa, Province of La Pampa, Argentina. To avoid alteration during transport, whey was stabilized with 0.1% (100 vol) hydrogen peroxide. In the laboratory, whey was heated to boiling point for 50 min to precipitate most of the protein, and then cooled and vacuum filtered. It was stored frozen to allow the availability of a raw material with constant composition. As whey provided by the manufacturing plant contained in the order of 40 g/l lactose, it was diluted with distilled water to give 30 g/l final concentration. Whey provided the sole carbon source.

Tests carried out in fermenters were performed at different agitation rates: 100, 200, 300, 400 and 500 rpm using a 1 L/L. min aeration rate. Equipment used was similar to a 5-liter New Brunswick unit, with two turbines, one as a foam break. Monitors were used to measure and control pH, temperature and agitation rates, measure dissolved oxygen partial pressure, and control foam by automatic addition of an antifoam silicone agent through a peristaltic pump. For tests with automatic pH control, a system operating with an Ingold sterilizable electrode and automatic addition of 5N sulphuric acid solution through peristaltic pumps was used. Controls were performed at different pH levels: 6.5, 7 and 7.5. The processes began at a pH value of 6.8.



Fig. 1 shows the results obtained in previous experiments carried out in rotary shaker (6). The medium contains lactose as carbon source and a similar medium where lactose is replaced by acid cheese whey (Medium 3, Table 1).


0005i02.gif (14097 bytes)

——— Lactose Medium
--------- Whey Medium
l Dry weight, o Lactose, O pH, D a-amylase activity.

Figure 1. Production of a-amylase from Bacillus Subtilis NRRL 3411 growing on lactose and whey medium in a rotary shaker. (250 rpm and 2.5 cm stroke). 


Production of a-amylase from this microorganism is associated with growth and reaches its maximun level during the stationary phase (5). Changes in the measured parameters followed a similar pattern reaching enzyme values of approximately 5500 DUN/ ml units. In all tests in which acid cheese whey was used, pH change was moderate with a tendency towards less alkaline values.

In both media, microorganisms grew as aggregates. Cell oxygen demand (maximum values) were in the order of 230 ml O2/L.h. On the basis of these results, all further tests were performed using cheese whey as carbon source.

Table 2 shows the results obtained at 100, 200, 300, 400 and 500 rpm in a mechanically stirred fermenter. The highest enzyme levels are obtained at 300 rpm. Here again, the microorganism develops as cell aggregates as in the stirred flasks. In general, the process was completed in less time using fermenters, with rapid change of pH, which becomes more noticeable as the agitation rate is increased.


0005i03.gif (19556 bytes)


At 100 and 200 rpm growth is lower, and in these conditions, for higher agitation rate, the maximum dry weight values are similar to those at 300, 400 and 500 rpm.

Table 3 shows oxygen dissolution values. It is evident that the value at 300 rpm is similar to that determined in the rotary shaker. Even though the values for dissolved oxygen increase from 400 rpm, enzyme concentrations do not behave likewise.


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On the basis of previous experiments, where it was shown that a similar concentration is reached in fermenters as in rotary shakers, and accounting for a rapid change of pH towards very alkaline values, new experiments were performed with a pH control system. pH control processes were carried out at 6.5, 7 and 7.5, reaching the highest enzyme concentrations at pH 7.5, (Fig 2), doubling the enzyme concentration and reaching a value of 10,000 DUN/ml. units. These tests were carried out at 300 rpm. Biomass concentration as well as lactose consumption throughout the process were similar to those for tests without pH control. Also, here the cell oxygen demand (maximum value) was in the order of 230 ml O2/L.h, (Fig. 3). The same cell aggregate type of growth is observed.


0005i05.gif (11705 bytes)

l Dry weight, o Lactose, O pH, D a-amylase activity, n Dissolved oxygen.

Figure 2. Production of a-Amylase from Bacillus Subtilis NRRL 3411 growing on whey medium in a mechanically stirred fermenter. (300 rpm and 1 L/L.min). 



0005i06.gif (11498 bytes)

l Dry weight, oLactose, O pH, D a-amylase activity, n Dissolved oxygen, O Cell oxygen demand.

Figure 3. Production of a-amylase from Bacillus Subtilis NRRL 3411 growing on whey medium in a mechanically stirred fermenter. (300 rpm and 1 L/L.min), with automatic pH (7.5) control.


The described results express the averages of triplicate experiments.



If we consider the results obtained in stirred Erlenmeyers, it becomes evident that whey can be used as source of lactose to obtain a-amylase. Media containing whey show lower changes in pH than those containing lactose. This behaviour can be attributed to the proteins contained in this by-product (10), namely lactoalbumin and lactoglobulin, which do not precipitate in the de-proteinization treatment which removes whey casein.

From fermentors results, it can be inferred that growth limitation at 100 and 200 rpm could be due to lack of oxygen, as shown by cell demand and oxygen dissolution values in Table 3. It can be observed that higher agitation rates produce higher biomass.

Even though it is necessary to supply oxygen for growth, an increase in stirring conditions allows for an improved enzyme production level up to 300 rpm. This behaviour could be related to different variables which can be considered separately. In general, it is observed that an increase in the agitation rate produces a shorter fermentation with a marked pH change towards alkaline values limiting enzyme production (4). An increase in agitation rate over 300 rpm changes the growth pattern of the cell aggregates. The decrease of enzyme levels at high stirring values could be related to enzyme denaturization (7) and to a change in the relationship O2- CO2 (9).

Tests carried out with automatic pH control indicate that a pH of 7.5 has a considerable influence on enzyme production, since double the a-amylase values were obtained for aeration conditions at low oxygen concentrations.

It can be concluded that, under conditions in which the highest enzyme concentration is obtained, pH plays a fundamental role in the accumulation of a-amylase. It is also worth mentioning that retaining the aggregated form of growth, possible up to 300 rpm, ensures a high production of a-amylase.



We thank the Consejo Nacional de Investigaciones Científicas y Técnicas (conicet) and Facultad de Ciencias Exactas y Naturales - UNLPam for financial support.




Produção de a-amilase em soro ácido de queijo como meio de cultura, com controle automático do pH.

O presente artigo teve o objetivo de relatar a produção de a-amilase usando uma estirpe de Bacillus subtilis NRRL 3411, usando soro de queijo como fonte de carbono. Foi determinada a influência da aeração do meio de cultura bem como o controle automático do pH. As determinações de a-amilase foram realizadas pelo método de DUN e a taxa de absorção de oxigênio em diferentes condições de aeração pelo método de Cooper. A demanda celular de oxigênio foi estabelecida em um respirômetro de Warburg e o nível de oxigênio dissolvido por meio de eletrodos galvânicos prata-chumbo. Os resultados indicam a possibilidade de uso do soro de queijo como fonte de carbono pois foram similares aos obtidos com lactose. Melhores rendimentos, 10.000 unidade DUN/ml, foram obtidos em 96 h de processo com aeração média -431 mLO2/l.h- e com controle operativo do pH a 7.5, condições estas que permitiram dobrar a atividade de amilase no caldo. Finalmente, foi observado que a obtenção de a-amilase está relacionada com a maneira de crescimento dos microrganismos nos agregados celulares.

Palavras-chave: Bacillus subtilis, soro ácido de queijo, controle automático do pH.




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* Corresponding author. Mailing address: Departamento de Química, Facultad de Ciencias Exactas y Naturales. UNLPam. Av. Uruguay Nro. 151. C.P. 6300. Santa Rosa, La Pampa, Argentina. Fax (02954) 432679. E-mail:

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