The influence of substrate source on the growth of Ralstonia eutropha, aiming at the production of polyhydroxyalkanoate

Abstract

With the aim of producing polyhydroxyalkanoates, a study of the influence of culture conditions (nitrogen and carbon sources and temperature) on the growth of Ralstonia eutropha in stirred flasks was carried out and the use of some low-cost sources (hydrolyzed lactose, inverted sugar and corn steep liquor) as evaluated. The best specific growth rate was obtained when inverted sugar was utilized as the substrate (mumax = 0.26 h-1). Two different phases in the assimilation of the carbon source were observed when hydrolyzed lactose was present, suggesting the assimilation first of glucose and then of galactose. To confirm the growth of Ralstonia eutropha using galactose as the only carbon source, experiments were carried out and the results showed that this bacterium is able to grow in the presence of this sugar at a growth rate of 0.13 h-1. The use of galactose by Ralstonia eutropha for its growth has not been reported in the literature until now. Corn steep liquor was found to be a viable alternative nitrogen source to ammonium sulfate. The results of experiments carried out at 30°C and 34°C were similar.

corn steep liquor; inverted sugar; polyhydroxyalkanoates; Ralstonia eutropha; specific growth rate

THE INFLUENCE OF SUBSTRATE SOURCE ON THE GROWTH OF Ralstonia eutropha, AIMING AT THE PRODUCTION OF POLYHYDROXYALKANOATE

C.Marangoni, A.Furigo Jr. and G.M.F.Aragão^*

Universidade Federal de Santa Catarina, UFSC,

Departamento de Engenharia Química e Engenharia de Alimentos,

Campus Universitário, Trindade, 88040-900, Florianópolis - SC, Brazil

Phone: [phone], Fax: [phone]

E-mail: [e-mail]

(Received: October 10, 2000 ; Accepted: May 21 2001)

Abstract - With the aim of producing polyhydroxyalkanoates, a study of the influence of culture conditions (nitrogen and carbon sources and temperature) on the growth of Ralstonia eutropha in stirred flasks was carried out and the use of some low-cost sources (hydrolyzed lactose, inverted sugar and corn steep liquor) as evaluated. The best specific growth rate was obtained when inverted sugar was utilized as the substrate (m_max = 0.26 h^-1). Two different phases in the assimilation of the carbon source were observed when hydrolyzed lactose was present, suggesting the assimilation first of glucose and then of galactose. To confirm the growth of Ralstonia eutropha using galactose as the only carbon source, experiments were carried out and the results showed that this bacterium is able to grow in the presence of this sugar at a growth rate of 0.13 h^-1. The use of galactose by Ralstonia eutropha for its growth has not been reported in the literature until now. Corn steep liquor was found to be a viable alternative nitrogen source to ammonium sulfate. The results of experiments carried out at 30°C and 34°C were similar.

Keywords: corn steep liquor, inverted sugar, polyhydroxyalkanoates, Ralstonia eutropha, specific growth rate

INTRODUCTION

Bioplastics can be used to substitute petrochemical plastics in the manufacture of many articles used as pack aging. Polyhydroxyalkanoates (PHA) are thermoplastic materials which fulfill this requirement. They are water insoluble polyesters synthesized by several bacteria for energy and an intracellular carbon storage, under limited growth conditions in excess carbon (Anderson and Dawes, 1990). Ralstonia eutropha is one of the most widely studied microorganisms with regard to PHA production due to the ease of accumulation of this polymer from renewable carbon sources (Doi, 1990). Poly (3-hydroxybutyrate) (P(3HB)) is a well-known PHA polymer synthesized by Ralstonia eutropha. Since these biopolymers have properties similar to those of petrochemical-derived polymers and offer the advantage of being biodegradable, optimization of their production on an industrial scale is a high priority (Byrom, 1992). PHA production in Ralstonia eutropha is carried out in two phases – a phase of unlimited growth, aiming at biomass generation, and another phase with nutrient (nitrogen, phosphate, oxygen, etc.) limitation, favoring polymer accumulation according to the source of carbon offered (Doi, 1990). Since PHA is accumulated as intracellular granules, it is important that the specific growth rate in the first phase be high, aiming at achieving optimal productivity of the final product. The main obstacle to industrial manufacture is the high production cost. Therefore, several studies have attempted to minimize production costs by for example, the use of low-cost carbon sources for bacterial growth or the development of new strains or more efficient techniques for polymer recovery (Byrom, 1992). Inverted sugar is available in Brazil as a low-cost substrate. Good results have been obtained using this substrate as the carbon source (Gomez et al., 1996). Corn steep liquor, an industrial waste, is a potentially useful substitute corn nitrogen source. Even after an essential pretreatment, this waste, still appears to be economically attractive, and studies have already shown satisfactory results for the product as a bacterial growth medium (Hoch, 1997). Whey, a dairy industry waste, is a low-cost alternative that can be used as the hydrolyzed lactose source (Wong and Lee, 1998). The objective of this work was to study the effect of temperature, the influence of nitrogen and carbon sources and the use of low-cost substrates on bacterial growth. Two nitrogen sources (ammonium sulfate and corn steep liquor), four carbon sources (glucose, fructose, inverted sugar and hydrolyzed lactose) and two temperatures (30^oC and 34^oC) were used.

MATERIAL AND METHODS

Microorganism and Culture Medium

A glucose-utilizing mutant of Ralstonia eutropha, DSM 545, was grown in nutrient broth (NB) medium containing 5.0 g l^-1 of meat peptone and 3.0 g l^-1 of meat extract. The mineral medium (MM) composition was the same as that used by Marangoni et al. (2000). After autoclaving the culture medium, a sterile phosphate solution was aseptically added to the culture medium to obtain a final concentration of 8.5 g l^-1 of Na₂PO₄.12H₂O and 1.5 g l^-1 of KH₂PO_4. In the experiments designed to study the influence of nitrogen source, 5 g l^-1 of (NH₄)₂SO₄ or 5 ml of pretreated corn steep liquor (corresponding to the same concentration of ammonium sulfate) was aseptically added to the medium. In the same way, the carbon source was added according to the experiment. Cultures were grown with only one carbon source and glucose, fructose or inverted sugar as aseptically added to obtain an initial concentration of 20 g l^-1.

Corn Steep Liquor

This substrate was obtained from Refinações de Milho do Brasil S.A.

The treatment carried out in corn steep liquor was as follows: first, a centrifugation step was carried out at 3000 min^-1 for 40 minutes. The pH was then adjusted to 7.0 with a solution of 7M NaOH. A further centrifugation under the same conditions was carried out and the liquor was sterilized by heating at 121^oC for 20 minutes. Centrifugation and sterilization were repeated to remove precipitated particles and the supernatant was placed on ice prior to use.

Lactose Hydrolysis

Lactose was hydrolyzed with Lactozim (Novo Nordisk). The conditions of lactose hydrolysis were: a substrate concentration of 50 g l^-1 of lactose, a pH of 6.8, a hydrolysis time of 90 min, a temperature of 40°C, an enzyme-to-substrate ratio of 0.08 g enzyme/g substrate

Hydrolysis was confirmed by glucose analysis with an enzymatic test and by the method of 3-5 dinitrosalicilic acid, which determines the concentration of reduced sugars.

Culture conditions

The experiments were carried out in stirred 1000 mL Erlenmeyer flasks containing 300 mL of medium, incubated at 150 rpm. The pH was initially adjusted to 7.0 by the addition of 5M KOH or 1M HCl. Samples were taken hourly for approximately 12 h corresponding to the growth phase. The temperature was 30°C in all experiments, except those where temperature was analyzed. In this case two temperatures were studied: 30 and 34°C. The inoculum concentration was approximately 0.5 g l^-1 of biomass.

Analytical Techniques

Total Biomass: cells were harvested by filtering a given volume (2 and 10 mL) of culture broth through preweighed polyamide membrane filters (0.2 mm pore size); they were them washed twice with distilled water and dried to constant weight at 100 ^oC. Cellular density was also determined by turbidimetric measurements at 600nm. The ratio between optical density and cellular concentration was determined by a standard curve of previous experiments.

RESULTS AND DISCUSSION

In the exponential growth phase, the specific growth rate and specific polymer production rate are constants. In this way, the specific growth rate can be represented by the total biomass.

Influence of Carbon Source

In the experiments performed, the maximum value for the specific growth rate obtained in inverted sugar (m_max = 0.26 h^-1) was a little higher than that obtained in glucose (m_max = 0.23 h^-1). This fact confirms that inverted sugar is a potential substitute for glucose, since its growth rate is higher and its cost is lower than those of glucose (Gomez et al., 1996). The value obtained for growth rate in glucose is similar to that reported by Kim et al. (1994). Growth in fructose showed a specific rate of 0.21 h^-1, a value that is a little lower than that obtained for glucose. It was expected that fructose would provide the best specific growth rate since in previous studies reported by Linko et al. (1993) this substrate presented the best results for cell productivity.

In the experiments with 25 g l^-1 of hydrolyzed lactose (corresponding to 12.5 g l^-1 of glucose and 12.5 g l^-1 of galactose), two phases, with different growth rates, were observed (Figure 1). The change in the specific growth rate occurs at around 5 h in culture. Analysis of the biomass concentration results shows that up until this moment approximately 1.2 g l^-1 of biomass was produced. According to the literature, the yield for R. eutropha is 0.5 g g^-1 (Aragão et al., 1996), and considering this biomass yield, 2.4 g l^-1 of glucose should have been consumed. Since the initial glucose concentration was 12.5 g l^-1, 10.1 g l^-1 of glucose should remain in the medium. This sugar concentration is near the critical concentration for cell growth in glucose (10 g l^-1; Oliveira, 1999). This fact suggests that the change in behavior is due to the consumption of another substrate (galactose) by the microorganism. The specific growth rates obtained for the first and second phases (Figure 1) were 0.20 h^-1 and 0.11 h^-1, respectively.

To confirm R. eutropha growth in galactose as the carbon source, one experiment was carried out with 0 g l^-1 of galactose as the only carbon source. The specific growth rate obtained for this bacterium was m_max = 0.13 h^-1 (Figure 2). The fact that this strain of R. eutropha can use galactose as the only carbon source has not been reported in the literature until now. Pries et al. (1990) reported the growth of R. eutropha in galactose, but the strain utilized was genetically modified with this aim in mind. The confirmation that R. eutropha can consume galactose is interesting because it means that both products of lactose hydrolysis (glucose and galactose) can be assimilated.

Our results are very close to those obtained when these sugars, glucose and galactose, are used as the only carbon source, confirming that consumption of glucose occurs initially, followed by consumption of galactose.

The best value for the specific growth rate was obtained when inverted sugar was used as the carbon source; we studied the effect of a mixture of glucose and fructose (sugars that are present in inverted sugar) in equal proportion and then compared this with the effect obtained with inverted sugar. The value of the maximum specific growth rate was 0.20 h^-1, lower than that obtained with inverted sugar (0.26 h^-1). This fact suggests that inverted sugar improves the growth rate, possibly due to its complex composition.

Influence of Temperature

The temperatures used in this study (30^oC and 34^oC) were based on those adopted in the literature (Aragão et al., 1996; Madden, 1998). The results of the experiments using 20 g l^-1 of hydrolyzed lactose as the carbon source at temperatures of 30^oC and 34^oC are shown in Table 1. The fact that we obtained similar results for the temperatures is interesting in view of industrial applications. Since the process is exothermic, utilization of higher temperatures (in this case, 34°C) is interesting since less energy is required to cool the fermentor.

Influence of Nitrogen Source

Table 2 shows the comparison of specific growth rates when ammonium sulfate and corn steep liquor were used as the nitrogen source with 20 g l^-1 of fructose and 40 g l^-1 of a mixed substrate (25% glucose, 25% fructose, 25% hydrolyzed lactose and 25% inverted sugar) as the carbon source.

It was observed that the experiments showed similar exponential behaviors and the values of the specific growth rates are very close. These results confirm that there are no substantial differences in the specific growth rate when corn steep liquor is used as a substitute for ammonium sulfate. Therefore, this industrial waste is an interesting and viable alternative to ammonium sulfate, although it is important to emphasize that this waste requires previous treatment.

CONCLUSIONS

Inverted sugar was the substrate which showed the highest maximum growth rate (m = 0.26 h^-1) of the carbon sources studied. The fact that Ralstonia eutropha can use galactose for its growth has not been reported in the literature until now. This is very interesting because it is another sugar from hydrolyzed lactose assimilated by the microorganism in substrates such as whey.

Corn steep liquor was substituted for ammonium sulfate, showing good results for specific growth rate.

The temperatures analyzed did not result in differences in the growth of the microorganism.

The use of low-cost substrates such as corn steep liquor, inverted sugar, and potentially, whey (hydrolyzed lactose) produced results comparable to those for the conventional sources, allowing a reduction in the processing of polyhydroxyalkanoate production cost.

ACKNOWLEDGEMENTS

Financial support was obtained from CNPq in the form of a fellowship for C. Marangoni.

NOMENCLATURE

m specific growth rate (h^-1); m_max maximum specific growth rate (h^-1); PHA Polyhydroxyalkanoate; P(3HB) Poly(3-hydroxybutyrate); DSM Deutsche Sammlung vor Mikrorganismen und Zellkulturen;

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*

To whom correspondence should be addressed

Publication Dates

• Publication in this collection
  06 Aug 2001
• Date of issue
  June 2001

History

• Accepted
  21 May 2001
• Received
  10 Oct 2000