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Sustain. Environ. Res., 25(2), 125-130 (2015)
125
Technical Note
Biological effect of magnetic field on the production of
polyhydroxyalkanoates
Marzie Fatehi and Seyed Ahmad Ataei*
Department of Chemical Engineering
Shahid Bahonar University
Kerman 7618891167, Iran
Key Words: Municipal wastewater, excess activated sludge, polyhydroxyalkanoates, magnetic field
ABSTRACT
This study reports on the effect of magnetic field at 0 to 50 mT on polyhydroxyalkanoate (PHA)
production in activated sludge. Tests were performed for excess carbon addition and PHA content and
some other parameters determined. In summary, this research indicated that higher PHA contents of 0.75
g L-1 were produced at magnetic field intensities of 5 and 20 mT, whereas the lowest PHA content was
recorded at 50 mT (0.55 g L-1). In addition, magnetic field influenced the type and amount of monomer
produced of the copolymer PHA. Higher amounts of butyrate and valerate monomer were observed at
magnetic field of 5 and 50 mT, respectively. Finally, in the present work, the magnetic field intensity
of 5 mT was determined as the optimum magnetic field by considering the ratio of PHA produced to
the amount of suspended solids in the activated sludge together with other economic factors (such as
costs related to the production of PHA, reduction of sludge volume and production of magnetic field).
INTRODUCTION
Biodegradable plastics are mainly constituted
of chemically or biologically produced polyesters.
Polyhydroxyalkanoates (PHAs) are natural macro
molecules of polyesters produced by a variety of
microorganisms and are currently considered as an
alternative to conventional plastics. PHAs are formed
from 3-hydroxy fatty acid monomers and accumulated
as a carbon/energy reserve source in microorganisms
[1-3].
More than 300 types of microorganism are able
to produce and store PHA under conditions of limited
nutrients and excess carbon source [1]. Bacteria can
synthesize a wide range of PHAs and approximately
150 different constituents of PHAs have been
identified. Currently polyhydroxybutyrate (PHB) and
copolymer P (HB/HV) are the only PHAs produced on
a commercial scale [4-6].
Over the past decades, the intrinsic resistance of
plastic materials to degradation has been increasingly
regarded as a source of environmental and waste
*Corresponding author
Email: [email protected]
management problems. Converting biodegradable
components in municipal sludge under thermophilic
conditions to volatile acids and further into PHAs
has its merits for sustainable development and waste
management such as less and safer sludge to be
handled, less methane produced in landfill sites, lower
cost for sludge disposal, if it can be partially utilized
as raw materials to produce valuable products, and
production of true biodegradable thermoplastics. Based
on previous studies, excess sludge volume can be
reduced to less than 30% by extracting of PHA from
activated sludge [7]. The idea of PHA production using
mixed culture arose from recognition of the PHA’s
role as a metabolic intermediate in microbial processes
for wastewater treatment (WWT). In most cases, a
mixed culture generates PHA from organic acids in
wastewater or organic acids that have been added from
other sources of industrial waste [8-10].
Magnetic field application is a new technique in
WWT for various objectives and its key advantages are
those of protein recovery, treatment of cells, simulation
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Fatehi and Ataei, Sustain. Environ. Res., 25(2), 125-130 (2015)
of enzymes and biological WWT. There have been
numerous studies on the effect of magnetic field on
microbial performance, but the results were usually
inconsistent. Some of studies showed a negative
effect, while most of them showed an enhancement in
growth, because the effect depended on the intensity of
magnetic field and the type of microorganisms. Yavuz
and Celebi indicated that a magnetic field tended to
increase bacterial activity and this effect was far more
noticeable in heterogeneous cultures (sewage) than
in pure culture [11]. Several other studies have been
conducted on the effect of magnetic field on chemical
oxygen demand (COD) removal from domestic and
industrial wastewater and they reported a strong
relationship between type of wastewater and intensity
of magnetic field [12-14]. To date, there has been no
serious investigation on effect of magnetic field on
PHA production, although magnetic field exposure has
definite positive and negative effects on the storage
capacity of PHA in microorganisms [10,13,15].
The effect of static magnetic field on production
of PHAs from different short-chain volatile fatty
acids (VFAs) by activated sludge process under the
aerobic dynamic feeding technique was investigated
in sequencing batch reactors with applied magnetic
field intensities of 7-42 mT [16]. These tests reported
that exposure to a static magnetic field had a definite
influence on biosynthesis of PHAs, and the effect was
dependent on field strength [16]. However, there have
been very few reports on changes of enzymatic activity
in cells and increased or decreased PHA production in
microorganisms subject to a magnetic field exposure.
The aim of the present research was to study the
effect of magnetic field on PHA content, amount and
type of monomer in the copolymer. An activated sludge
from Kerman municipal wastewater plant in Kerman,
Iran, was transferred into a PHA production reactor
under magnetic field at the various strengths of 0
(control sample), 5, 10, 15, 20, 25 and 50 mT.
MATERIALS AND METHODS
1.Batch Reactor Experiment
Figure 1 shows a 1.5 L cylindrical plexi reactor
with 1 L working volume. The reactor was fed with
sodium acetate as the sole carbon source at 3000 mg
L -1. Oxygen was supplied by an air compressor at
the rate 1 L min-1. The reactor was operated at 15 ±
1 °C without pH control. Samples were taken from
the sludge in the batch reactor at regular intervals,
and analyzed for PHA, biochemical oxygen demand
(BOD), pH and mixed liquor suspended solids (MLSS).
The initial characterization of the activated sludge,
Fig. 1. PHA production reactor.
from Kerman municipal wastewater plant, Kerman,
Iran is presented in Table 1.
2.Magnetic Field Exposure
The reactor was operated under seven different
intensities of magnetic field (0-50 mT). The magnetic
field was generated by magnets and intensity of the
magnetic field was produced at the center of reactor.
The magnetic field intensity was measured by a Tesla
meter placed in the middle of the reactor.
3.Analysis
PHA measurement was measured according to the
method described in Ataei et al. [17]. 5 mL of samples
were centrifuged at 6000 rpm and 30 min. Then 2
mL of chloroform and 1 mL of acidified methanol
containing benzoic acid as the internal standard were
added to the sludge. Samples were heated for 2 h at
100 °C by COD reactor (model WTW) then cooled
rapidly. One mL of distilled water was added to the
solution and vortexed for 1 min. Organic and aqueous
layers were allowed to separate. The bottom organic
phase (methyl ester of alkanoates) was transferred
into a fresh tube. Then 2 μL of this phase was injected
into a gas chromatograph (model Varian CP 3800) at
Table 1. Characterization of activated sludge
Characterization
C:N:P
pH
COD (ppm)
BOD (ppm)
Sludge volume index
Volatile suspended solids/
Total suspended solids
MLSS (g L-1)
Range
24:0.14:1
7.65
5490
1290
90-95
0.75
3600
Fatehi and Ataei, Sustain. Environ. Res., 25(2), 125-130 (2015)
250 °C, which was equipped with a flame ionization
detector and column (Capillary 8 CP, 30 m x 1 μm).
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 temperature
was increased to 150 °C at a rate of 25 °C min-1 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). BOD was
measured by the Standard Method [18].
127
sodium acetate to the activated sludge as a carbon
source. In the PHA production process, biodegradable
organic material and excess carbon source are
converted into VFAs. These VFAs are then consumed
by microorganisms to produce PHA. Figure 2 shows
the changes in BOD with time; reduction of 69% BOD
at 30 h cultivation.
2.PHA Production with Magnetic Field
The changes in BOD and PAH levels during the
course of 48 h cultivation are shown in Fig. 2. The PAH
content increased with time with the highest amount
of PHA content (0.6 g L-1) at 30 h. Thereafter (30-48
h), the PHA production declined due to less substrate
available with cells undergoing endogenous phase [19].
Therefore, a longer aeration time may select a microbial
community with lower PHA production capacity
than that selected under shorter aeration time [20]. At
aeration time lower than 30 h, the system may be at the
log-growth phase. There is an excess amount of food
available during this period and the microorganism
population is less than that during the stationary phase.
As a result, an activated sludge processes operating in
the stationary phase can produce more PHAs compared
to productivity in endogenous or log-growth phases.
Based on these results, optimum aeration time for PHA
production for this system was determined to be 30 h.
Evaluations for BOD of activated sludge were
taken before and after additions of sodium acetate as
excess carbon source, these were 1290 and 6110 ppm,
respectively.
The amount of carbon can be increased by adding
In the second step of experiments, the influence
of magnetic field on the production of PHAs was
investigated at 30 h cultivation (same operating
condition) and results of these tests were compared
with those of the control sample (without magnetic
field).
Figure 3 plots show that magnetic field exposure
influenced PHA production by the activated sludge.
Results shown in Fig. 3 clearly demonstrate that
magnetic field intensities of 5, 10, 15 and 20 mT had
positive effects on PHA production, while intensities
of 25 and 50 mT had negative effects. There are a
number of possible causes for the effect of magnetic
exposure on PHA production, and its influence on
substrate consumption and enzyme activity [11,21].
Xu et al. indicated that magnetic exposure can change
rate of biological reactions [15]. The first probable
reason for decreased PHA production, especially at 10
and 15 mT compared to 5 and 20 mT, is that magnetic
field enhanced the rate of consumption of the substrate
resulting in cells entering into the death phase earlier
than would be expected (30 h).
The second reason is that magnetic field perhaps
influences enzyme activity as reported previously [15].
Some studies have confirmed the effect of exposure
to magnetic field on amplification pattern of microbes
in the sludge [10]. At magnetic field intensities of 5
and 20 mT, enzymatic activity may be improved and
PHA production increased. However, detailed study is
Fig. 2. Effect of cultivation time on the PHA content (∆)
and BOD changes (◊).
Fig. 3. Effect of magnetic field on amount of PHA production.
RESULTS AND DISCUSSION
1.PHA Production without Magnetic Field (Control
Sample)
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Fatehi and Ataei, Sustain. Environ. Res., 25(2), 125-130 (2015)
required to further investigate this mechanism.
Finally, a comparison was made between results
of this part and those results described in others [16] as
shown in Fig. 4. For magnetic field intensity lower than
21 mT, it had a positive influence on PHA produced
by mixed culture. The result reported in Li et al. [22]
demonstrate that magnetic field intensity of 25 mT had
a positive effect on algal cultivation while this intensity
had negative effect on mixed culture (activated sludge).
PHB and PHV biosynthetic processes are quite
similar but different enzymes activate each process.
Previous researches have confirmed the effect of
magnetic field on enzymatic activity [11,23,24]. Results
of the present research show that variations of mass
percentage of HB and HV are related to intensity of
magnetic field (Fig. 5). The maximum HB occurred at
5 mT at 81% with the minimum of HB observed at 50
mT.
The magnetic field intensity of 50 mT had a
negative effect on PHA production (reduced to 8%
PHA compared to the control sample). Considering
that the purpose was to generate the maximum amount
of HV in the copolymer, the negative effect of this
magnetic field can be ignored. Due to the effect of
HV or HB on mechanical properties of biopolymer
and according to our aim of producing PHA (increase
production or improve properties of the biopolymer) it
is preferable to apply the magnetic field intensity with
optimum effect.
Figure 6 shows the PHA/MLSS ratio at different
magnetic field intensities after 30 h cultivation in
1 L of sample. If PHA production and biomass
volume reduction are the aims of process, it would be
appropriate to determine a high ratio of PHA/MLSS.
The highest ratio reached at 25 mT, but PHA content
at in this magnetic field (0.58 g L-1) was less than that
determined at the magnetic field intensity of 20 mT
(0.75 g L-1). Consequently, according to PHA content
and PHA/MLSS ratio, intensity of 20 mT was more
efficient than that of 25 mT. In the other tests, PHA
contents at 5 and 20 mT were approximately similar,
while energy used to generate magnetic field intensity
of 5 mT is much less than that for 20 mT. In general,
the costs of PHA production and generation of a
magnetic field have higher costs than that needed for
volume reduction. Then magnetic field intensity of 5
mT was determined as the optimum intensity.
Finally, it is suggested that according to economic
factors such as cost of PHA production, generation
of magnetic field and reduction of sludge volume, the
most efficient intensity of magnetic field needs to be
selected.
CONCLUSIONS
Fig. 4. Percentage of PHA production compared to control sample in this study and previous research [16].
The main results can be summarized as follows:
• The best position to optimize PHA production is in
the stationary phase.
• The highest amount of PHA production was 0.6 g L-1
after 30 h aeration.
• BOD removal during the PHA production process
after 30 h aeration was 69%.
• Magnetic field intensity less than 20 mT increased
production of PHA (positive effect) and higher than
Fig. 5. Mass percentage of HB and HV in the copolymer
PHA.
Fig. 6. Ratio of PHA/MLSS at different magnetic field
intensity.
Fatehi and Ataei, Sustain. Environ. Res., 25(2), 125-130 (2015)
20 mT it had a negative effect on PHA production.
• These results demonstrate that exposure to magnetic
field had a definite influence on PHA biosynthesis
and the effect was dependent on field strength.
The maximum and minimum PHA contents were
observed at 20 and 50 mT to be 0.75 and 0.55 g L-1,
respectively.
• Magnetic field influenced on mass percentages of
HB and HV in the copolymer PHA. Higher amounts
of HV and HB in the copolymer occurred at 50 and
5 mT, respectively.
• Magnetic field intensity of 5 mT was determined as
the most efficient intensity when economic factors
were also taken into consideration.
ACKNOWLEDGEMENT
Authors gratefully acknowledge Amir Sadeghi
Pour Marvi from Shahid Bahonar University, Kerman,
Iran for critical reading of the manuscript.
REFERENCES
1. Chee, J.Y., S.S. Yoga, N.S. Lau, S.C. Ling,
R.M.M. Abed and K. Sudesh, Bacterially produced
polyhydroxyalkanoate (PHA): Converting
renewable resources into bioplastics. In: A.
Méndez-Vilas (Ed.). Current Research, Technology
and Education Topics in Applied Microbiology
and Microbial Biotechnology. Formatex Research
Center, Badajoz, Spain (2010).
2. N a i k , S . , S . K . V. G o p a l a n d P. S o m a l ,
Bioproduction of polyhydroxyalkanoates from
bacteria: A metabolic approach. World J. Microb.
Biot., 24(10), 2307-2314 (2008).
3. Khanna, S. and A.K. Srivastava, Recent advances
in microbial polyhydroxyalkanoates. Process
Biochem., 40(2), 607-619 (2005).
4. Suriyamongkol, P., R. Weselake, S. Narine, M.
Moloney and S. Shah, Biotechnological approaches
for the production of polyhydroxyalkanoates in
microorganisms and plants - A review. Biotechnol.
Adv., 25(2), 148-175 (2007).
5. Brandl, H., R.A. Gross, R.W. Lenz and R.C. Fuller,
Plastics from bacteria and for bacteria: Poly(βhydroxyalkanoates) as natural, biocompatible, and
biodegradable polyesters. Microb. Bioprod., 41,
77-93 (1990).
6. Finch, C.A., Chemistry and Technology of
Biodegradable Polymers. Chapman & Hall,
London, UK (1995).
7. Lee, S. and J. Yu, Production of biodegradable
thermoplastics from municipal sludge by a twostage bioprocess. Resour. Conserv. Recy., 19(3),
129
151-164 (1997).
8. Tian, P.Y., L.A. Shang, H. Ren, Y. Mi, D.D. Fan and
M. Jiang, Biosynthesis of polyhydroxyalkanoates:
Current research and development. Afr. J.
Biotechnol., 8(5), 709-714 (2009).
9. Dionisi, D., M. Majone, V. Papa and M. Beccari,
Biodegradable polymers from organic acids by
using activated sludge enriched by aerobic periodic
feeding. Biotechnol. Bioeng., 85(6), 569-579
(2004).
10. Chen, H., H.B. Li and Y.F. Xia, Acclimating PHA
storage capacity of activated sludge with static
magnetic fields. Enzyme Microb. Tech., 46(7),
594-597 (2010).
11. Yavuz, H. and S.S. Celebi, Effects of magnetic
field on activity of activated sludge in wastewater
treatment. Enzyme Microb. Tech., 26(1), 22-27
(2000).
12. Hattori, S., M. Watanabe, T. Endo, H. Togii and
K. Sasaki, Effects of an external magnetic field
on the sedimentation of activated sludge. World J.
Microb. Biot., 17(3), 279-285 (2001).
13. Jagadeesh, A., Investigation of the Effect
of Magnetic Field on the Chemical Oxygen
Demand Removal of Wastewater. Master Thesis,
Department of Chemical Engineering, Ryerson
University, Toronto, Canada (2006).
14. K r z e m i e n i e w s k i , M . , M . D e b o w s k i , W.
Janczukowicz and J. Pesta, Effect of sludge
conditioning by chemical methods with magnetic
field application. Pol. J. Environ. Stud., 12(5), 595605 (2003).
15. Xu, Z.L., H. Chen, H.Y. Wu and L.X.Y. Li, 7 mT
static magnetic exposure enhanced synthesis of
poly-3-hydroxybutyrate by activated sludge at
low temperature and high acetate concentration.
Process Saf. Environ., 88(4), 292-296 (2010).
16. Chen, H. and X.L. Li, Effect of static magnetic
field on synthesis of polyhydroxyalkanoates from
different short-chain fatty acids by activated
sludge. Bioresource Technol., 99(13), 5538-5544
(2008).
17. Ataei, S.A., E. Vasheghani-Farahani, S.A.
Shojaosadati and H.A. Tehrani, Isolation of
PHA-producing bacteria from date syrup waste.
Macromol. Symp., 269(1), 11-16 (2008).
18. Hach, C.C., R.L. Klein Jr. and C.R. Gibbs,
Introduction to Biochemical Oxygen Demand.
Hach Company, Loveland, CO (1997).
19. Metcalf and Eddy, Wastewater Engineering:
Treatment and Reuse. McGraw-Hill, New York
(2003).
20. Mokhtarani, N., Determination of Effective Factors
on the Production of Poly-Hydroxyalkanoates by
130
Fatehi and Ataei, Sustain. Environ. Res., 25(2), 125-130 (2015)
Activated Sludge. Ph.D. Dissertation, Department
of Environmental Engineering, Tarbiat Modares
University, Tehran, Iran (2005).
21. Berlot, M., T. Rehar, D. Fefer and M. Berovic, The
influence of treatment of Saccharomyces cerevisiae
inoculum with a magnetic field on subsequent
grape must fermentation. Chem. Biochem. Eng. Q.,
27(4), 423-429 (2013).
22. Li, Z.Y., S.Y. Guo, L. Li and M.Y. Cai, Effects of
electromagnetic field on the batch cultivation and
nutritional composition of Spirulina platensis in
an air-lift photobioreactor. Bioresource Technol.,
98(3), 700-705 (2007).
23. Jung, J.T. and S. Sofer, Enhancement of phenol
biodegradation by south magnetic field exposure. J.
Chem. Technol. Biot., 70(3), 299-303 (1997).
24. Blanchard, J.P. and C.F. Blackman, Clarification
and application of an ion parametric resonance
model for magnetic field interactions with
biological systems. Bioelectromagnetics, 15(3),
217-238 (1994).
Discussions of this paper may appear in the discussion section of a future issue. All discussions should
be submitted to the Editor-in-Chief within six months
of publication.
Manuscript Received: February 5, 2014
Revision Received: July 16, 2014
and Accepted: August 18, 2014