Influence of Magnetic Field on Polyhydroxyalkanoate Production by Activated Sludge

Polyhydroxyalkanoates (PHAs) are naturally occurring polyester that can be accumulated in bacterial cells as a carbon and energy storage compound. They are metabolized by many microorganisms. These biopolymers accumulate in microbial cells under stress conditions such as limitation of nitrogen, phosphate, sulfur, oxygen, and excess carbon source. Currently, PHAs have attracted increasing interest as promising alternatives to conventional plastics. This is due to their biodegradability capability in being produced from renewable resources.
The major barrier to wide application of PHAs is their current high cost. Biological waste water treatment is in wide use in the world, and produced large amounts of activated sludge that requires some way of disposal. Moreover, some organisms in activated sludge are known that they have ability to accumulate PHA. Consequently, it is intuitively obvious that production of PHAs from excess activated sludge would be a beneficial economical process.
A number of studies have been carried out on influence of magnetic field on bacterial activity, removal of chemical oxygen demand (COD), sedimentation of activated sludge and etc. Some of these studies indicated that a magnetic field tended to increase bacterial activity and this effect was far more noticeable in heterogeneous culture (waste water) than in pure culture. But, the results of all researches about influence of magnetic field on microorganism performance were inconsistent. Some of them showed negative and positive effects on PHA production. One of them indicated that positive and negative effect of magnetic field on PHA production depended on the intensity of magnetic field. In this study, we attempted to show the effects of different intensities of magnetic field on PHA production by activated sludge. We studied influence of magnetic field with intensity of 0, 5, 10, 15, 20, 25, and 50 mT.
Material and methods
Seven batch reactors, with individual working volume of 1 L were used for this experiment. In these systems, excess activated sludge, with sludge retention time (SRT) of 5 days, was transferred into the reactors. Activated sludge was collected from municipal waste water plant in Kerman City, Iran. Sewage cannot produce the polymer alone, upon activated sludge was fed with sodium acetate (as excess carbon source), with concentration of 3000 mg L^-1, at a level. Reactors were aerated with air compressors and oxygen rate was 1L min^-1. The activated sludge in batch reactors were sampled at regular intervals and the amounts of PHA were determined. The reactors were operated under the seven different magnetic fields, without pH and temperature control. The magnetic fields were generated by magnets and their intensities were measured by Tesla meter.
For Measuring of PHA, 5mL of samples were centrifuged at 6000 rpm in 30 min. Then 2 mL of chloroform and 1 mL of acidified methanol containing benzoic acid as the internal standard was added to the deposit sludge. Samples were heated for 2 hours at 100˚C by COD reactor (model WTW). After cooling, 1mL of distilled water was added and they were shaken for 1 min to separate phases. Then, 2 μL of bottom phase was injected into gas chromatograph (model Varian CP 3800) at 250˚C, which was equipped with a flame ionization detector (FID) and column (Capilary cp – sil 8 cp, 30m×1μm). The detector temperature was 280 ˚C. Helium was used as the carrier gas. Initial oven temperature was 80˚C which was held constant for 1 min. Then, the temperature was increased to 150˚C at a rate of 25˚C/min and retained for 1 min. Calibrations of PHA were done with a standard poly (3- hydroxybutyric- co- 3- hydroxyvaleric acid) (12 wt% PHV)(Sigma, USA).
Discussion and result
Access of microorganisms to oxygen can be an obstacle for PHA production. Therefore, in order to achieve ideal aeration time for control sample, activated sludge was sampled in defined times during 48 hours for measurement of PHA. The results of this step showed that increasing aeration time causes food restriction on the microorganism and conditions are provided to produce more polymers. The highest amount of PHA was 0.6 g.L^-1. This occurred after 30 hours of aeration. After this time, microorganism entered into death phase and microbial population was changed. Thus, the best position to PHA production is stationary phase. In other words, activated sludge in this phase produce bio polymer more than death phase.
We investigated influence of magnetic field intensity of 5, 10, 15, 20, 25, and 50 mT on PHA production. The results of this study were simultaneously compared with the results of PHA production without the magnetic field (control sample). These results demonstrated that the magnetic exposure had definitely influenced the PHA content and it was depended on magnetic field intensity. The maximum PHA content reached 0.75 gL^-1 at 20 mT. This was 25% higher than that of the control and the minimum occurred at 50 mT, which is equivalent to 0.55 gL^-1 (Fig. 1).
 
 
Figure 1. Influence of magnetic field on PHA production
 
In the general case, in this research, magnetic field intensities was less than 20 mT and increased PHA content (positive effect). The magnetic field intensity higher than 20 mT had negative effects on PHA production. The reason of this inconsistent is the effect of magnetic field on enzyme behavioral change in microorganism's cell, microorganism's performance, bacterial activity, substrate consumption rate, lipid solubility of the substrate in cell membrane and etc.
The results also showed that magnetic field influenced type and amount of monomers in copolymer PHA. Type and amount of monomers were studied at 20 and 50 mT. Since, biosynthesis of polyhydroxybutyrate (PHB) and polyhdroxyvalerate (PHV) are similar together but with deferent enzymes. The magnetic field may accelerate electron motion during enzymatic processes. The result of this is an increase in the activity of some enzymes. At 20 and 50 mT, amount of HB were 74 and 24% and the amount of HV were 26 and 76%, respectively (Fig. 2).
PHB and PHV biosynthetic processes are quite similar together, but they are done by different enzymes. In some previous researches, effect of magnetic field on activity of enzyme and enzyme reaction has been confirmed. Blanchard and Blackman certified that magnetic field influenced on performance of enzymes and their potential. On the other hand, there were always unpaired electrons during in biological process of enzymatic reactions, where exposure to magnetic field could influence the reaction by changing the electrons spin state. The influence of magnetic field on enzymes will change the reaction.
 
 
Figure 2. Type and amount of monomers in copolymer PHA
 
Conclusion
According to the magnetic field effect on the type and amount of monomers in copolymer PHA and the aims of using magnetic field (increasing production of copolymer PHA in comparison with the control sample or increasing production of specific monomer in copolymer, regardless their positive and negative effect on PHA production) recommended that the effect of magnetic field was investigated for every culture separately.