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Enzyme and Microbial Technology 46 (2010) 594–597

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

cclimating PHA storage capacity of activated sludge with static magnetic fields

hen Honga,∗, Li HaiBob, Xia YunFenga

Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310027, ChinaZhejiang Forestry Academy, Hangzhou 310023, Zhejiang, China

r t i c l e i n f o

rticle history:eceived 31 December 2009

a b s t r a c t

The effect of a static magnetic field on the acclimation of activated sludge with high polyhydroxyalkanoate(PHA) storage capacity was evaluated under aerobic dynamic feeding (ADF) conditions. The acclimation

eceived in revised form 5 March 2010ccepted 8 March 2010

eywords:HAsctivated sludge

processes were carried out in Sequence Batch Reactors (SBRs) with static magnetic field intensities of42, 21, 7, or 0 mT. Static magnetic exposure significantly influenced the PHA accumulation in activatedsludge. PHA content in biomass reached 66% (on a COD basis) at a magnetic field intensity of 21 mT and anorganic load of 5.42 g L−1, which was 16% higher than that of the control. Analysis by PCR-DGGE showedthat the underlying mechanism might involve a change in the dominant species of microorganism in

to the

tatic magnetic fieldcclimation

each reactor in response

. Introduction

Polyhydroxyalkanoates (PHAs) are biological polyesters pro-uced by a wide variety of microorganisms as a form of intracellulararbon and energy storage material [1]. PHAs have recentlyttracted the attention of industrial scientists because of theirotential use as practical biodegradable and biocompatible plasticaterials with properties ranging from thermoplasts to elastomers

2].Biological wastewater treatment is in wide use in the world, and

roduces large amounts of excess activated sludge, which requiresome form of disposal [3]. Consequently, it is intuitively obvioushat producing PHAs from this excess activated sludge would be aighly economical process. It would not only decrease PHA produc-ion cost, but would also have the benefit of reducing the amountf excess activated sludge that requires disposal. Although a num-er of the organisms found in activated sludge are known to havehe ability to accumulate PHAs, the actual amounts of PHAs foundn excess activated sludge are very low [4]. However, acclimatizinghe sludge is one means of improving PHA production.

Aerobic dynamic feeding (ADF) is one of the most promisingechniques for improving the efficiency of PHA accumulation inctivated sludge [5]. A number of studies have been carried out

n PHA production with activated sludge using the ADF technique6–10]. Dionisi et al. [11] reported that a maximum polymer frac-ion of 50% (on a COD basis) in the biomass could be obtained usingctivated sludge enriched by ADF. Bengtsson et al. [1] showed 48%

∗ Corresponding author. Tel.: +86 571 8795 2560; fax: +86 571 8797 7703.E-mail address: chen hong@zju.edu.cn (C. Hong).

141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2010.03.004

static magnetic field.© 2010 Elsevier Inc. All rights reserved.

PHA content in sludge (dry weight) from a full-scale municipalwastewater treatment plant.

Yavuz and Celebi [12] indicated that a magnetic field tended toincrease bacterial activity and this effect was far more noticeable inheterogeneous cultures (sewage) than in pure culture. The couplingeffect of static magnetic exposure and ADF on the microorganismsresponsible for PHAs production has not yet been seriously investi-gated. In addition, only a few studies have incorporated moleculartechniques in investigations into PHA production [13]. This kind ofmolecular method could also be applied to characterize the PHAproducing microorganism communities in activated sludge accli-mated under the influence of magnetic fields.

In the present research, we have attempted to show the effectsof applied magnetic fields on the acclimation of activated sludgewith high PHA storage capacity under ADF. The excess activatedsludge, taken from a municipal wastewater treatment plant, wasinoculated into four Sequence Batch Reactors (SBRs) exposed to dif-ferent intensities of static magnetic fields. We also took molecularsnapshots of the microbial populations of these activated sludgesunder magnetic fields using PCR-DGGE technology.

2. Materials and methods

2.1. Acclimation process

Four SBRs, with individual working volumes of 1 L, were operated under fourdifferent magnetic field intensities, i.e., 42, 21, 7, and 0 mT. The activated sludge,taken from the Sibao municipal wastewater treatment plant, Hangzhou, China, was

aerated for 4–5 days to exhaust the initial substrate and then diluted to 1.2 g L−1

Mixed Liquor Suspended Solids (MLSS). The diluted activated sludge was then inoc-ulated into the SBRs for acclimation with mixtures of acetate, propionate, andbutyrate using the ADF technique. The start-up organic load (measured by COD) was360 mg L−1 and was increased gradually to 720 mg L−1, 1450 mg L−1, 2170 mg L−1,3240 mg L−1, 4362 mg L−1, 5420 mg L−1, and 6480 mg L−1 over 45 days [14].

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a bright main band of approximately 1500 bp in length that wasamplified from the cultivated sludge from the different reactors.The appearance of one main band suggested that only one micro-bial population was present in the sludge under different externalmagnetic fields.

C. Hong et al. / Enzyme and Mic

The SBR system was operated as follows: 10 min of feeding, then the “feast”tage (according to the removal time of the substrate), followed by a “famine” stage,nd last 2 min of final sludge withdrawal. The ratio of “feast”:“famine” time wasontrolled at 1:3. Oxygen was supplied by an air compressor and was passed throughceramic membrane disperser installed in the bottom of each reactor, to maintain

he dissolved oxygen (DO) concentration at around 80% of the saturation value. Theeactor was operated at 25 ± 1 ◦C without pH control.

The SBR feed, ratio of C:N:P, mineral media and the SBR operation were per-ormed as described previously [15]. 20 mg L−1 thiourea was also added to inhibititrification. The pH value of the mineral solution was adjusted to 7.0 prior to

eeding.

.2. Analytical techniques

The sludge in the batch reactor was sampled at regular intervals. Acetate,utyrate, propionate were measured in filtered samples (0.45-�m porosity)y gas chromatography (Kexiao, GC-1690) equipped with a GDX-401 col-mn (length: 0.5 m, diameter: 3 mm) and a flame ionization detector (FID),orresponding organic acids of analytical agent quality grade are standardaterials. PHA (PHB, PHV) determination was performed as described pre-

iously [15]. Calibrations of PHB and PHV were done with a standard poly3-hydroxybutyric-co-3-hydroxyvaleric acid) (12% of the monomer 3-HV) of naturalrigin (Sigma–Aldrich).

The intensity of the magnetic field was measured by a Teslameter (Model 6010,ell Technologies Inc.) placed in the middle of the reactor. The dry weight of biomassas measured as Volatile Suspended Solids (VSS), according to standard methods.H of samples was measured with a pH meter (PHS-3C).

DNA extraction, PCR amplification and sequence determination of 16S rDNA.otal microbial community DNA was extracted from the sample according to previ-usly published protocols [16]. The extracted DNA was amplified by PCR using therimer set of 27F and 1492R [17]. PCR amplification was carried out in a 20 �L vol-me of reaction mixture containing 75 ng of genomic DNA, 1× PCR buffer, 2.5 mMgCl2, 250 �M of each dNTP, 0.5 �M of each primer, 1.5 U of Taq DNA polymeraseith a DNA thermal cycler (TC-XP, Hangzhou Bioer, China) using the followingarameters: 5 min at 94 ◦C, followed by 35 cycles of 1 min at 94 ◦C, 1 min at 55 ◦C,min at 72 ◦C, and a final extension cycle of 7 min at 72 ◦C. The amplified 16S rDNA-CR products were excised from the gels with a BioSpin Gel Extraction Kit (Hangzhouioer, China). The sequences were determined using the purified PCR products andhe two primers (BcaBEST RV-M and BcaBEST M13-47) with a ABI 377 automatedNA sequencer.

.3. Phylogenetic analysis

Phylogenetic analysis was based on distance methods using MEGA version 3.118]. Distances were calculated based on pairwise deletion of gaps and missingata between taxa without correction of P values. Phylogenetic trees were gener-ted using a neighbor-joining tree-building algorithm [19]. Confidence in branchingoints was determined by bootstrap analysis (1000 replicates) [20].

.4. Calculations

The amounts of substrate, PHAs, and biomass were all converted into COD units.mounts of PHAs were calculated as percentage of Volatile Suspended Solids (VSS)n a mass basis (PHAs% = g CODPHAs/g CODVSS × 100).

. Results and discussion

.1. SBR reactor performance

The initial parameters of activated sludge prior to acclimationere pH 5.26, VSS 1.2 g L−1 and a 0.17% of PHAs content in biomass

on COD basis). The value of pH increased with increases in organicoad and then stabilized between pH 8.40–9.20 during the acclima-ion (data not shown). Among the four SBRs, pH was higher in the1 and 7 mT reactors and lower in the 0 mT reactor for each organic

oad. The time of substrate removal (feast phase) was the same forll SBRs at lower organic load, but changed with the increase inrganic load. The substrate removal time for the 21 mT reactor waslways the shortest at higher organic load.

Changes in PHA content in the biomass of the four SBRs as the

rganic load increased is shown in Fig. 1. In general, PHA contentn the biomass of each SBR increased with increasing organic loadmeasured by COD) from 0.36 g L−1 to 5.42 g L−1, but then decreasedeyond this level. Peak values of 66% for PHA content occurred at1 mT at an organic load rate of 5.42 g L−1, it was 63% at 7 mT, and

Fig. 1. PHAs content in biomass with organic load increasing under different mag-netic field intensities ((�) 42 mT, (�) 21 mT, (�) 7 mT, and (�) 0 mT).

the lowest value of 46% was seen at 42 mT. In contrast, the highestPHA content in the control reactor was 50%.

Based on these results, magnetic field intensities of 21 and 7 mTappeared to be suitable for enhancing the PHA storage capacity ofactivated sludge. In contrast, an intensity of 42 mT was inhibitory,suggesting that magnetic fields had both positive and negativeeffects on the PHA storage ability of microorganisms derived fromactivated sludge. Li et al. [21] reported that EMF (electric magneticfield) had a double-sided effect on Spirulina platensis cultivation.The intensity of 0.25 T EMF was found to be suitable for the algalcultivation and the maximum cell dry weight increased by 22% ina time period 2 days less than that of the control.

3.2. 16S rDNA-PCR

The 16S rDNA fragment amplification of 4 microflora sam-ples collected from the reactors was performed using one pair ofprimers, 27F and 1492R. The electrophoretogram in Fig. 2 shows

Fig. 2. 16S rDNA amplification pattern of microbes in the sludge under differentmagnetic field intensities. Lane M was DNA marker (DL2000 bp); lanes 1–4 repre-sented the microflora sample Ba1–Ba4, which were collected from the 42, 21, 7, and0 mT reactors.

596 C. Hong et al. / Enzyme and Microbial Technology 46 (2010) 594–597

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ig. 3. Neighbor-joining (NJ) phylogenetic tree of four Bacillus species and their relatairwise deletion, bootstrap = 1000; the numbers in each node represented bootstrand marked with asterisks were collected and examined in this study, and others o

.3. Analysis of 16S rDNA-sequences

The four collected sludge samples from the reactors were iden-ified primarily as Bacillus species by microscopic examination. The6S rDNA region sequences were determined and compared withach other. The PCR fragments of the 16S rDNA from the 4 collectedamples ranged from 1444 to 1509 bp in length. The G + C contentf the 16S rDNA of the 4 samples ranged from 50.54% to 57.13%.here were greater differences in the length of 16S rDNA betweena2 and Ba1–Ba3–Ba4 and in the G + C contents of 16S rDNA amonga1, Ba3 and Ba2–Ba4, which suggested that there were differencesmong the microbial populations in the collected sludge samplesnder different external magnetic fields.

.4. Phylogenetic analysis

19 sequences of 16S rDNA region including the sequences of themicrobial species in this study and 15 closely related species or

trains retrieved from GenBank-database were used for phyloge-etic analysis. After excluding the unalienable regions of the 5′ endnd 3′ end, the final alignment with a total 1560 position was usedo construct a phylogenetic tree. The phylogenetic tree presentedn Fig. 3 shows that 19 sequences were grouped into two distin-uishable clades. The 15 strains including Ba1, Ba2 and Ba4 in this

tudy formed a unique, weakly supported Clade 1, while the otherstrains including Ba3 in this study were clustered into another

lade 2 with a 100% bootstrap value.The results of phylogenetic analysis indicated that Ba1 and Ba2

n this study may be a species of Flavobacterium and Clostridium

ains/species based on 16S rDNA region sequences. Nucleotide: Kimura 2-parameter,port value, and the numbers lower than 50 were not shown. The 4 samples in boldd from GenBank.

respectively, and that both Ba3 and Ba4 in this study were uncertainBacillus strains. The various Bacillus species in the collected sludgesamples suggested a drastic change in the microbial populationsunder different external magnetic fields.

4. Conclusions

Microorganisms with high PHA storage ability could be acclima-tized by gradually increasing organic load under ADF condition withthe appropriate range of static magnetic field intensity, whereasthe magnetic field intensity of 42 mT was inhibiting. Our studyindicated the dominant microorganisms collected from each reac-tor were different. Based on this result, it could be inferred thatthe microflora of all reactors might be affected by magnetic fields,which resulted in changes in the predominant microflora, therebybringing about changes in the abilities to produce PHAs in the reac-tors.

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