WO2000052189A1

WO2000052189A1 - Method for the production of polyhydroxyalkanoate

Info

Publication number: WO2000052189A1
Application number: PCT/NL2000/000143
Authority: WO
Inventors: Marinus Cornelis Maria Van Loosdrecht
Original assignee: Technische Universiteit Delft
Priority date: 1999-03-03
Filing date: 2000-03-03
Publication date: 2000-09-08
Also published as: AU2949800A; NL1011431C2

Abstract

The invention relates to a method for the production of polyhydroxyalkanoate, for example from a waste stream. In a first step of this method, microorganisms are grown and in a second step polyhydroxyalkanoate is accumulated. According to the invention, the microorganisms are in a first step subjected to a selection, which selection comprises a first phase in which substrate is added, alternating with a second phase in which substrate is withheld. After at least two selection cycles, selected microorganisms are used in the second step for the accumulation of polyhydroxyalkanoate.

Description

Method for the production of polyhydroxyal anoa'lj'e

The present invention relates to a met]bό$ for the production of polyhydroxyalkanoate, wherein i) in a first step, microorganisms are grown in a culture medium in the presence of substrate; and ii) in a second step, with the supply of an oxygen-comprising gas and substrate, the microorganisms accumulate polyhydroxyalkanoate .

Such a method is generally known in the art . For example, Haase-Aschoff, I. (Stuttg. Ber. Siedlungswasser- wirtsch. 91, p. 232 (1985)) describes a method wherein microorganisms grow on waste water, yielding active sludge, wherein the supply of an excessive amount of substrate results in the formation of polybetahydroxybutyric acid, a bacterial reserve lipid.

The object of the present invention is to provide a more efficient method of producing polyhydroxyalkanoate, wherein the method may be carried out under non-sterile conditions. Another object is to provide a method capable of using a waste stream or a technically pure substrate, yielding a valuable polyhydroxyalkanoate .

To this end the method according to the invention is characterized in that in the first step the microorganisms are subjected to a selection while being supplied with an oxygen-comprising gas, which selection comprises a first phase in which substrate is added, alternating with a second phase in which substrate is withheld, the first phase comprising a period during which an excessive amount of substrate is present in the culture medium, and the second phase comprising a period during which the substrate concentration is less than 0.20 g of substrate in relation to the carbon content per litre, wherein after at least two selection cycles selected microorganisms are used in the second step for the accumulation of polyhy- droxyalkanoate . Since the object with water purification is to limit the amount of sludge as much as possible, active sludge comprises organisms having a low conversion rate. Surprisingly it has been found, that under the conditions of the method according to the invention in which organisms having a high growth rate are selected, the organisms that in addition to polyhydroxyalkanoate store also poly- phosphate or glycogen as reserve, do not compete very well with the organisms that produce polyhydroxyalkanoate . Thus with the method according to the invention, it is possible to obtain a higher yield of polyhydroxyalkanoate. During part of the second phase, the substrate concentration is preferably lower than 0.1 g/1, more prefer-ably lower than 0.03 g/1 and most preferably lower than 0.01 g/1. In order to attain such a low-substrate phase, the supply of substrate is simply discontinued after the first phase. Within the context of the invention the term "to withhold" substrate also includes supplying substrate in amounts small enough to meet the respective conditions. Conven- iently, the substrate is available in a waste stream, such as substrate-rich percolate water from a rubbish dump, or process liquid.

According to a suitable embodiment, some of the selected microorganisms are used in the second step and unused microorganisms continue to grow in the first step under the selection conditions.

This means, that by not each time departing from active sludge as source of suitable microorganisms, the earlier selection of suitable microorganisms is fully utilized.

Preferably the substrate in the second step is a fatty acid-comprising stream.

Fatty acids are very suitable starting materials for forming polyhydroxyalkanoate . According to a preferred embodiment, the substrate in the first step is also a fatty acid-comprising stream. In such a case, the metabolism of the microorganisms to be used in the second step is already optimal for forming polyhydroxyalkanoate.

According to a favourable embodiment, the fatty acid-comprising stream is obtained by under controlled conditions fermenting a carbohydrate substrate.

This increases the application possibilities of the method according to the present invention. Suitably, the carbohydrate is of a technical quality, or it may be available in the form of process water, for example, from the sugar industry.

Advantageously, the first step is carried out in a first reactor and the second step in a second reactor.

Such a set-up simplifies the process control. According to a preferred embodiment, the second step is carried out such that at least one element chosen from N, P, and S causes a limitation.

Such a limitation restricts the growth of the organisms in the second step and further increases the yield of polyhydroxyalkanoate.

The present invention will now be elucidated with reference to an exemplary embodiment and with reference to the drawing in which Figure 1 shows a graph of two substrate concentrations, the ammonium concentration and the biomass formed during a selection cycle of 6 hours;

Figure 2 is a graphical representation of the concentration of polyhydroxybutyrate and polyhydroxyva- lerate during a selection cycle;

Figure 3 represent the concentration and the polyhydroxybutyrate content in an accumulation reactor; and to

Figure 4 represents an electron micrograph of bacteria comprising PHB granules selected by means of a selection reactor, . EXAMPLE

A selection reactor having a volume of 2 litres was continuously mixed and aerated with 2.28 1/min com- pressed air. By means of a thermostatic bath the medium in the selection reactor was maintained at 34 °C. The pH of the medium was maintained that 7.0 with the aid of a potassiumhydroxyde solution.

The selection reactor was operated in cycles, one cycle taking 6 hours. Each cycle started with a bacteria- containing volume of one litre. To this, mineral medium was added in 2 minutes (1.4 g/1 of ammonium chloride, 0.2 g/1 of magnesium sulphate and 5 ml/1 of a standard trace element solution) . Further, during the first hour of the cycle 100 ml of concentrated fatty acid solution were added (160 g/1 of acetate; 12.4 g/1 of propionate) . At t=4 hours 6.4 g of potassium dihydrogenphosphate were fed to the system. At t=5h45 1 litre of the bacteria suspension was discharged in 15 minutes to an accumulation reactor. This leaves 1 litre of bacteria-containing medium with which the next selection cycle is started. At the start, the medium was inoculated with active sludge. The entire operation is performed under non-sterile conditions, that is to say that the medium and compressed air were not sterilized. Such an open microbial culture means a considerable saving in costs compared with other production processes that are carried out under sterile conditions for the protection of pure cultures. The saving in costs relates both to capital outlay (sterilization apparatus) and direct costs (energy) .

By operating the selection reactor according to the method of the invention, a bacteria culture is obtained that is capable of quickly taking up a lot of fatty acid and to convert them into a copolymer of β- hydroxybutyrate and β-hydroxyvalerate. After a number of cycles (after the start-up with active sludge approximately 10 cycles) a steady state is reached in which each cycle is basically identical with the preceding one. Such a cycle is represented in Figure 1. It can be seen that the concentration of biomass increases during the first 1.5 hours, and then remains practically constant. As described above, at the end of the cycle the bacteria suspension obtained in the selection reactor is fed to an accumulation reactor. This reactor was operated at the same pH and temperature as the selection reactor, and in the same manner continuously aerated and mixed. After the addition of the bacteria suspension, ammonium, magnesium sulphate and trace elements were dosed up to the same content and amount (0.9 1) as at the beginning, of the cycle in the selection reactor. Then acetate was fed for 12 hours two the accumulation reactor. The acetate was partly used for growth but primarily for the formation of poly-β-hydroxybutyrate. The curve of the concentration in the PHB-content in the accumulation reactor is shown in Figure 3. Figure 4 shows an electron micrograph of the bacteria obtained, in which the PHB granules are visible.

Claims

1. A method for the production of polyhydroxyalkanoate, wherein i) in a first step, microorganisms are grown in a culture medium in the presence of substrate; and ii) in a second step, with the supply of an oxygen-comprising gas and substrate, the microorganisms accumulate polyhydroxyalkanoate, characterized in that in the first step the microorganisms are subjected to a selection while being supplied with an oxygen-comprising gas, which selection comprises a first phase in which substrate is added, alternating with a second phase in which substrate is withheld, the first phase comprising a period during which an excessive amount of substrate is present in the culture medium, and the second phase comprising a period during which the substrate concentration is less than 0.20 g of substrate in relation to the carbon content per litre, wherein after at least two selection cycles selected microorganisms are used in the second step for the accumulation of polyhydroxyalkanoate.

2. A method according to claim 2, characterized in that some of the selected microorganisms are used in the second step and unused microorganisms continue to grow in the first step under the selection conditions.

3. A method according to claim 1 or 2 , character- ized in that the substrate in the second step is a fatty acid-comprising stream.

4. A method according to claim 3, characterized in that the substrate in the first step is a fatty acid- comprising stream.

5. A method according to claim 4, characterized in that the fatty acid-comprising stream is obtained by under controlled conditions fermenting a carbohydrate substrate.

6. A method according to one of the preceding claims, characterized in that the first step is carried out in a first reactor and the second step in a second reactor.

7. A method according to one of the preceding claims, characterized in that the second step is carried out limiting at least one element chosen from N, P, and S